CA2609980A1 - Treatment of protein folding disorders - Google Patents

Abstract

In certain embodiments, the invention is directed to a method for treating a protein folding disorder such as Alzheimer's disease, dementia, Parkinson's disease, Huntington's disease and prion-based spongiform encephalopathy. The method comprises the administration to a subject of a compound of the formula (I) wherein A and B are independently a mono- or bicyclic aromatic group or heteroaromatic cyclic group. In preferred embodiments, the compounds are bis-indole compounds.

Description

TREATMENT OF PROTEIN FOLDING DISORDERS

BACKGROUND OF THE INVENTION

[0001] Protein folding disorders include neurodegenerative conditions such as, e.g, Alzheimer's disease, dementia, Huntington's disease, Parkinson's disease and prion-based spongiform encephalopathy (e.g., Creutzfeldt-Jakob disease).

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease which first manifests with mild cognitive, memory and behavioral symptoms that gradually worsen in severity and eventually lead to dementia. It is the most common cause of dementia, accounting for between 42 and 81% of cases, as determined in various studies (Nussbaum, RL; Ellis, CE. NEnglJMed, 2003, 348: 1356-64). It affects 2.5 % of people 65-74 years of age, 4% of people aged 75-79, 11 % of those aged 80-84, and 24% of those aged 85-93 years (Siegel, GJ; Agranoff, BW; Albers, RW;
Molinoff, PB, Basic Neurochemistry. Fifth ed. 1994, New York: Raven Press, pp). Accounting for 100,000 deaths annually in North America alone, AD is the fourth leading cause of death in industrialized societies, preceded only by heart disease, cancer and stroke (Schenk, DB; Rydel, RE; May, P; Little, S; Panetta, J;
Lieberburg, I; Sinha, S. JMed Chem, 1995, 38: 4141-54). AD affects individuals in all races and ethnic groups, occurring slightly more commonly in females than males.

[0003] There is no remission in the progression of Alzheimer's disease, nor are there any disease-stabilizing drugs currently available (Selkoe, DJ; Schenk, D. Annu Rev Pharmacol Toxicol, 2003, 43: 545-84). As such, onset of the disease is inevitably followed by increasing mental and physical incapacitation, loss of independent living, institutionalization and death. There is usually an 8-10 year period from symptom onset until death, but patients can survive for 20 years or more after the initial diagnosis of AD is made (Siegel).

[0004] Accordingly, there exists a need in the art for an agent which can be used for the treatment of Alzheimer's disease and other protein folding disorders.

[0005] All documents referred to herein are incorporated by reference in their entireties for all purposes.

OBJECTS AND SUMMARY OF THE INVENTION

[0006] It is an object of the present invention to provide compounds and methods for treating protein folding disorders.

[0007] It is an object of certain embodiments of the present invention to provide compounds and methods for treating neurodegenerative diseases such as, e.g., Alzheimer's disease, tauopathies, cerebral amyloid angiopathy, Lewy body diseases (e.g. Parkinson's disease), dementia, tauopathies, cereberal amyloid angiopathies, Huntington's disease and prion-based spongiform encephalopathy.

[0008] It is an object of certain embodiments of the present invention to provide compounds and methods for treating systemic amyloidoses such as, e.g., secondary systemic amyloidosis, particularly those affecting the peripheral nerves, spleen, kidney, heart, intestine, smooth muscle or pancreas.

[0009] It is an object of the present invention to provide pharmaceutical compositions comprising an effective amount of a compound for treating protein folding disorders.

[0010] It is an object of certain embodiments of the present invention to provide pharmaceutical compositions comprising an effective amount of a compound for treating neurodegenerative diseases such as, e.g., Alzheimer's disease, tauopathies, cerebral amyloid angiopathy, Lewy body diseases (e.g. Parkinson's disease), dementia, Huntington's disease, prion-based spongiform encephalopathy and a combination thereof.

[0011] It is an object of certain embodiments of the present invention to provide pharmaceutical compositions comprising an effective amount of a compound for treating systemic amyloidoses, particularly those affecting the peripheral nerves, spleen, kidney, heart, intestine, smooth muscle or pancreas.

100121 It is an object of certain embodiments of the present invention to provide compounds, methods and pharmaceutical compositions for inhibiting tau protein aggregation in a subject or patient.

[0013] Other objects and advantages of the present invention will become apparent from the disclosure herein.

[0014] In certain embodiments, the present invention is directed to a method for treating a protein folding disorder comprising administering an effective amount of a compound of formula (I) to a patient in need thereof:

Ri R2 .~~~ 'n A B

(I) [0015] wherein A and B are independently a mono-, bi- or tri-cyclic aromatic or heteroaromatic substituent; wherein n=0 or 1; wherein, when n=1, R' and R2 are independently hydrogen, alkyl, cycloalkyl, alkoxy, hydroxy, halogen, aryl, or together represent the group =0; wherein A is substituted by A'(,) and B is substituted by B1(y) wherein x and y are independently an integer from 0 to 4; and A](X) and Bl(y) are each independently, for each value of x and y, selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl;
alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, thio, thioether, cyano, nitro, halogen, carboxylic acid and sulfonic acid;

or a pharmaceutically acceptable salt thereof.

[0016] In certain preferred embodiments, Al(x) and B1(y) are each independently, for each value of x and y, selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, thio, thioether, cyano, nitro, halogen or a pharmaceutically acceptable salt thereof.
[0017] In certain embodiments, the invention is directed to a method for treating a protein folding disorder comprising administering a compound of formula (1) to a subject wherein the subject is treated for the protein folding disorder.

[0018] In certain embodiments of the disclosed method, A and B of formula (I) are independently selected from the group consisting of phenyl, pyridyl, pyrrolyl, thiophenyl, furanyl, triazolyl, indolyl, naphthyl, benzofuranyl, quinolinyl, isoquinolinyl, benzothiophenyl, benzooxazolyl and benzimidazolyl.

[0019] In certain embodiments of the disclosed method, at least one of A and B
of formula (I) are indolyl and in certain embodiments, both of A and B of formula (I) are indolyl.

[0020] In certain embodiments of the disclosed method, the compound of formula (I) is:

Al (x) /B1 (Y) J-N
3 Ra (IA) [0021] In certain embodiments of the disclosed method, the compound of formula (I) is:

~ i A ()\ / R~ RZ B (Y) I I
N N
R 3 1a (IB) [0022] In certain embodiments of the disclosed method, the compound of formula (I) is:

A () -~ R' R2 \ / N
N

R3 B (r) (IC) [0023] In certain embodiments of the disclosed method, x is 1 and A' is at the 5, 6 or 7 position.

[0024] In certain embodiments of the disclosed method, x is 1 and A' is COzH.

[0025] In certain embodiments of the disclosed method, x is 1 and A' is at the position; wherein R' and R2 are independently hydrogen, alkyl, cycloalkyl, alkoxy, hydroxy, halogen, or aryl.

[0026] In certain embodiments of the disclosed method, x is 1 and A' is COZH
and is at the 5 position.

[0027] In certain embodiments of the disclosed method, x is 1 and A' is at the position; wherein R' and R 2 are independently hydrogen, alkyl, alkoxy, hydroxy or halogen.

[0028] In certain embodiments of the disclosed method, R3 and R4 are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, arylalkyl, alkylcarbonyl, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, arylsulfonyl or alkylsulfonyl.

[0029] In certain embodiments of the disclosed method, x is 1 and A' is CO2H
and is at the 6 position.

[0030] In certain embodiments of the disclosed method, A' is at the 5 position.
[0031] In certain embodiments, A' is hydroxy.

[0032] In certain embodiments of the disclosed method, A' is selected from the group consisting of halogen, hydroxy, alkyl, alkoxy, aryl and heteroaryl.

[0033] In certain embodiments of the disclosed method, in Bl is at the 5 or 6 position.
[0034] In certain embodiments of the disclosed method, Bl is selected from the group consisting of halogen, hydroxy, alkyl, alkoxy, aryl, thio, thioether, and trihalomethoxy.

[0035] In certain embodiments of the disclosed method, B1 is at the 5 position.

[0036] In certain embodiments of the disclosed method, B' is selected from the group consisting of halogen, hydroxy, alkyl, alkoxy, aryl and heteroaryl.

[0037] In certain embodiments of the disclosed method, Bl is at the 7 position.

[0038] In certain embodiments of the disclosed method, y is 1 and B1 is at the position; wherein R' and R2 are independently hydrogen, alkyl, cycloalkyl, alkoxy, hydroxy, halogen, or aryl.

[0039] In certain embodiments of the disclosed method, y is I and Bi is CO2H.
[0040] In certain embodiments of the disclosed method, n is an integer from 1 to 10;
wherein, when n is not 0, R' and RZ are substituted on one or more carbons and is as described above.

[0041] In certain embodiments of the disclosed method, the compound of formula (1) is selected from the group consisting of:

3,3'-bi-indolyl;
5-methoxy-3-(5-methoxyindol-3-yl)-indole;
3-(5-bromoindol-3-yl)-indole-5-carboxylic acid;
3-(indol-3-yl)-indole-5-carboxylic acid;
3-(5-methoxyindol-3-yl)-indole-5-carboxylic acid;
3-(5-fluoroindol-3-yl)-indole-5-carboxylic acid;
3-(5-chloroindol-3-yl)-indole-5-carboxylic acid;
3-(5-methylindol-3-yl)-indole-5-carboxylic acid;
3-(5-(trifluoromethoxy)-indol-3-yl)indole-5-carboxylic acid;
3-(5-hydroxyindol-3-yl)-indole-5-carboxylic acid;
3-(indol-3-yl)-indole-6-carboxylic acid;
3-(indol-3-yl)-indole-7-carboxylic acid;
3-(5-hydroxyindol-3-yl)-indol-5-ol; and a pharmaceutically acceptable salt thereof.

[0042] In certain embodiments of the disclosed method, the compound of formula (I) is selected from the group consisting of:
di-(indol-3-yl)methane;
3-(( indol-l-yl)methyl)-indole;
bis-(5-methoxy-indol-3 -yl)methane;

5-methoxy-3 -((5-methoxy-indol-l-yl)methyl)-indole;
3-((indol-3-yl)methyl)-indole-5-carboxylic acid;
bis-(indole-5-carboxylic acid-3-yl)methane;
bis-(5-hydroxy-indol-3 -yI)methane;
1,2-di-(indol-3-yl)ethane;

3-(2-(5-bromo-indol-3-yl)ethyl)-indole-5-carboxylic acid;
1,2-bis-(indole-5-carboxylic acid-3-yl)ethane; and a pharmaceutically acceptable salt thereof.

[0043] In certain embodiments the present invention is directed to a method for treating a protein folding disorder comprising administering an effective amount of a compound of fonnula (II) to a patient in need thereof:

R (ql) Ra N

I R I R(q2) N
1 , R

(II) wherein q, and q2 are each independently selected from an integer from 0 to 4;
wherein each R' and each R 2 is independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy; cycloalkyloxy, trihalomethoxy, aryloxy, arylcarbonyl, alkoxycarbonyl, amino, hydroxy, thioether, cyano, nitro, halogen, and carboxylic acid; and wherein R3, R4, R5, R8 and R9 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, cycloalkyloxy, trihalomethoxy, aryloxy, arylcarbonyl, alkoxycarbonyl, amino, hydroxy, thioether, cyano, nitro, halogen, and carboxylic acid; and wherein R6 and R7 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, arylalkyl, alkylcarbonyl, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, arylsulfonyl, alkylsulfonyl;

or pharmaceutically acceptable salts thereof.

[0044] In certain preferred embodiments, R' and R2 are the same and are hydroxy.
[0045] In certain preferred embodiments, R3, R4, and R5 are each alkyl.

[0046] In certain embodiments the present invention is directed to a method for treating a protein folding disorder comprising administering an effective amount of a compound of formula (III) to a patient in need thereof Rs 1 I Ra R(4') I R11 N
N

1Rio 2 R7 R (q2) R~Z

R5 Rs (III) wherein q, and q2 are each independently selected from an integer from 0 to 4;
wherein each R' and each R2 is independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, cycloalkyloxy, trihalomethoxy, aryloxy, arylcarbonyl, alkoxycarbonyl, amino, hydroxy, thioether, cyano, nitro, halogen, and carboxylic acid; and wherein R3, R4, R5, R6, R9, R10, R" and R12 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, cycloalkyloxy, trihalomethoxy, aryloxy, arylcarbonyl, alkoxycarbonyl, amino, hydroxy, thioether, cyano, nitro, halogen, and carboxylic acid; and wherein R7 and R8 are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, arylalkyl, alkylcarbonyl, arylcarbonyl;
alkoxycarbonyl, aryloxycarbonyl, arylsulfonyl, alkylsulfonyl;

or pharmaceutically acceptable salts thereof.

[0047] In certain preferred embodiments, R' and R2 are the same and are hydroxy.

[0048] In certain preferred embodiments, R3, R4, R5, and R6 are each alkyl.
[0049] In certain embodiments the present invention is directed to a method for treating a protein folding disorder comprising administering an effective amount of a compound of formula (IV) to a patient in need thereof:

N (a2) (e (q1) /N R N
/ Rg 1 4 R7 RS R

wherein q, and qz are each independently selected from an integer from 0 to 4;
wherein each R' and each R3 are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, cycloalkoxy, trihalomethoxy, aryloxy, arylcarbonyl, alkoxycarbonyl, amino, hydroxy, thioether, cyano, nitro, halogen, and carboxylic acid;

wherein R2 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl and cycloalkynyl;

wherein R4 and R9 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl and -R8; wherein R8 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, arylalkyl, arylcarbonyl, alkoxycarbonyl, amino; and wherein R5, R6 and R7 are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, arylalkyl, alkylcarbonyl, arylcarbonyl;
alkoxycarbonyl, aryloxycarbonyl, arylsulfonyl, alkylsulfonyl; or pharmaceutically acceptable salts thereof, isomers, stereoisomers, or diastereomers thereof.

[0050] In certain preferred embodiments, R8 is benzyl.

[0051] In certain preferred embodiments, R4 is hydrogen or carbobenzyloxy.
[0052] In certain embodiments, the invention is directed to a method for treating a protein folding disorder comprising administering a compound of formula (IV) to a subject wherein the subject is treated for the protein folding disorder.

[0053] In certain embodiments, of the disclosed method, the compound of formula (IV) is selected from the group consisting of CBZ-L-Trp-L-Trp-OH;
CBZ-L-Trp-D-Trp-OH;
CBZ-D-Trp-L-Trp-OH;
CBZ-D-Trp-D-Trp-OH;
H-L-Trp-L-Trp-OH;
H-L-Trp-D-Trp-OH;
H-D-Trp-L-Trp-OH;
H-D-Trp-D-Trp-OH;
and pharmaceutically acceptable salts thereof.

[0054] For purposes of the present invention "CBZ" means carbobenzyloxy.

[0055] In certain embodiments the present invention is directed to a method for treating a protein folding disorder comprising administering an effective amount a compound of formula (V) to a patient in need thereof:

~N O Rs N

R1(4l N R3(q2) N

R O
(V) wherein q, and q2 are each independently selected from an integer from 0 to 4;
wherein each R' and R3 are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, cycloalkoxy, trihalomethoxy, aryloxy, arylcarbonyl, alkoxycarbonyl, amino, hydroxy, thioether, cyano, nitro, halogen, and carboxylic acid; and wherein R4, R5, R6 and R7 are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, arylalkyl, alkylcarbonyl, arylcarbonyl;
alkoxycarbonyl, aryloxycarbonyl, arylsulfonyl, alkylsulfonyl; or pharmaceutically acceptable salts thereof, isomers, stereoisomers, or diastereomers thereof.

[0056] In certain embodiments, the invention is directed to a method for treating a protein folding disorder comprising administering a compound of formula (V) to a subject wherein the subject is treated for the protein folding disorder.

[0057] In certain embodiments, of the disclosed method, the compound of formula (V) is selected from the group consisting of Cyclo(L-Trp-L-Trp);
Meso-cyclo(Trp-Trp);
Cyclo(D-Trp-D-Trp); and pharmaceutically acceptable salts thereof.

[0058] In certain embodiments, the present invention is directed to a method for treating a protein folding disorder comprising administering an effective amount of a compound of formula (VI) to a patient in need thereof:

~Q)n \

A' , ' R (q) (VI) wherein A is a mono-, bicyclic, or tricyclic aromatic or heteroaromatic ring structure;

Q is -C-, -CH-, or -CH2-n is an integer from 0 to 4;
q is an integer from I to 3;

R' is alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, alkylcarbonyl, alkoxy, cycloalkyloxy, trihalomethoxy, aryloxy, arylcarbonyl, alkoxycarbonyl, amino, hydroxy, thioether, cyano, nitro, halogen, carboxylic acid, a mono-, bicyclic, tricyclic aromatic or heteroaromatic ring; wherein when R' is a mono-, bicyclic, tricyclic aromatic or heteroaromatic ring, then R' is optionally further substituted with one or more R2 groups each independently selected from the group consisting of alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, hydroxy, cycloalkyloxy, trihalomethoxy, aryloxy, arylcarbonyl, alkoxy;

wherein A is further optionally substituted with one or more R3 groups independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, cycloalkyloxy, trihalomethoxy, aryloxy, arylcarbonyl, alkoxycarbonyl, amino, hydroxy, thioether, cyano, nitro, halogen, and carboxy; or pharmaceutically acceptable salts thereof; and wherein Formula (VI) is not an unsubstituted or substituted indole-3-propionic acid.

[0059] In certain embodiments, A is selected from the group consisting of indoly], phenyl, pyridyl, pyrrolyl, thiophenyl, furanyl, tetrazolyl, naphthyl, benzofuranyl, quinolinyl, and isoquinolyl.

[0060] In certain embodiments the present invention is directed to a method for treating a protein folding disorder comprising administering an effective amount of a compound of formula (VII) to a patient in need thereof (ql) Rl (ql) N
N N
I3 l4 (VII) wherein ql and qz are each independently selected from an integer from 0 to 4;
wherein each R' and each R 2 is independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, cycloalkyloxy, trihalomethoxy, aryloxy, arylcarbonyl, alkoxycarbonyl, amino, hydroxy, methoxy, thioether, cyano, nitro, halogen, and carboxylic acid; and wherein R3 and R4 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, arylalkyl, alkylcarbonyl, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, arylsulfonyl or alkylsulfonyl;

or pharmaceutically acceptable salts thereof.

[0061] In certain preferred embodiments, the 2,3 bond of aza-indole is reduced.
[0062] In certain embodiments, the compounds are borane adducts at N-7 of the aza-indole.

[0063] In certain preferred embodiments, the compound of formula (VII) is selected from the group consisting of 3-(5-methoxy-indol-3-yl)-7-azaindole; 3-(5-bromo-indol-3-yl)-7-aza-indole; 3-(7-aza-indol-3-yl)-indol-5-ol; 3-(2,3-dihydro-7-aza-indol-3-yl)-indol-5-ol; 3-(7-aza-indol-3-yl)-indole-5-carboxylic acid; and pharmaceutically acceptable salts thereof.

[0064] In certain embodiments the present invention is directed to a method for treating a protein folding disorder comprising administering an effective amount of a compound of formula (VIII) to a patient in need thereof:

R 1 (q1) Al \ / \

X

(VIIl) wherein Al and A2 are independently selected from the group consisting of hydrogen, any substituted or non-substituted aromatic ring, carboxylic acid;
and pharmaceutically acceptable salts thereof;

wherein X is selected from the group consisting of oxygen, sulfur or N-R2, where R2 is selected from the group consisting of hydrogen, alkyl, aryl, sulfonylaryl, t-butoxycarbonyl (tBOC); and wherein R' is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, cycloalkyloxy, trihalomethoxy, aryloxy, arylcarbonyl, alkoxycarbonyl, amino, hydroxy, methoxy, thioether, cyano, nitro, halogen, carboxylic acid; and pharmaceutically acceptable salts thereof.

[0065] In certain preferred embodiments, the compound of formula (VIII) is selected from the group consisting of 2,3-bis(4-methoxybenzyl)-indole-5-carboxylic acid; 2,3-bis(4-hydroxybenzyl)-indole-5-carboxylic acid; 3-(4-hydroxybenzyl)-indole-5-carboxylic acid; and pharmaceutically acceptable salts thereof.

[0066] In certain embodiments, the invention is directed to compounds of formula (I), (II), (III), (IV), (V), (VI), (VII) or (VIII).

[0067] In certain embodiments, the present invention is directed to a compound of formula (IX):

A B

(IX) wherein A and B are indolyl substituents; wherein A is substituted by A' and B is substituted by B'(y); wherein x and y are independently an integer from 0 to 3 and the sum of x and y is from 1 to 3; and each A' and each B' are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, thio, thioether, cyano, nitro, halogen, carboxylic acid and sulfonic acid;

or a pharmaceutically acceptable salt thereof.

[0068] In certain embodiments, the present invention is directed to a compound of formula (IX):

A B
(IX) wherein A and B are indolyl substituents; wherein A is substituted by A' and B is substituted by B' C,); wherein x and y are independently an integer from 0 to 4 and the sum of x and y is at least 1;

each A' and each B' are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, thio, thioether, cyano, nitro, halogen, carboxylic acid and sulfonic acid;
or a pharmaceutically acceptable salt thereof.

[0069] In certain embodiments, the present invention is directed to a compound of formula (IX):

A B
(IX) wherein A and B are indolyl substituents; wherein A is substituted by A'(,q and B is substituted by B1 (y); wherein x and y are independently an integer from 0 to 4 and the sum of x and y is at least 1;

each A' and each Bl are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, thio, thioether, cyano, nitro, halogen, carboxylic acid and sulfonic acid; provided that the total number of hydroxy substituents is less than 4;

or a pharmaceutically acceptable salt thereof.

[0070] In certain embodiments of the present invention, the point of attachment for at least one of A and B is at the 1, 2 or 3 position of the indolyl.

[0071] In certain embodiments of the present invention, the point of attachment for both A and B is at the 1, 2 or 3 position of the indolyl.

[0072] In certain embodiments of the present invention, A is substituted by A'(,) in at least one of the 4, 5, 6 or 7 positions.

[0073] In certain embodiments of the present invention, B is substituted by Bl(Y) in at least one of the 4, 5, 6 or 7 positions.

[0074] In certain embodiments of the present invention, the compound of formula (IX) is:

A'(X) -~ B'(Y) If N N
\R2 (IXA) [0075] In certain embodiments of the present invention, x is 1 and A' is at the 5 position.

[0076] In certain embodiments of the present invention, y is I and B1 is CO2H.
[0077] In certain embodiments of the present invention, Bl is at the 5, 6 or 7 position.
[0078] In certain embodiments of the present invention, Al is selected from the group consisting of halogen, OC1_3 alkyl and OC(halogen)3.

[0079] In certain embodiments of the present invention, x is 0, y is 1 and Bl is COzH
at the 5, 6 or 7 position.

[0080] In certain embodiemtns of the present invention, R' and R2 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, arylalkyl, alkylcarbonyl, arylcarbonyl;

alkoxycarbonyl, aryloxycarbonyl, arylsulfonyl and alkylsulfonyl.

(0081] In certain embodiments, the present invention is directed to a pharmaceutical composition comprising an effective amount of a compound of formula (IX):

A B
(IX) wherein A and B are indolyl substituents; wherein A is substituted by A'(,) and B is substituted by B1 (y); wherein x and y are independently an integer from 0 to 4;
and each AI and each B' are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, thio, thioether, cyano, nitro, halogen, carboxylic acid and sulfonic acid;

or a pharmaceutically acceptable salt thereof to treat a protein folding disorder.
[0082] In certain embodiments, the present invention is directed to a pharmaceutical composition comprising an effective amount of a compound of formula (IX):

A B
(IX) wherein A and B are indolyl substituents; wherein A is substituted by A'(X) and B is substituted by B'(y); wherein x and y are independently an integer from 0 to 4;
and each A' and each B' are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, thio, thioether, cyano, nitro, halogen, carboxylic acid and sulfonic acid;

or a pharmaceutically acceptable salt thereof;

to treat a protein folding disorder, e.g., a neurodegenerative disease such as Alzheimer's disease, tauopathies, cerebral amyloid angiopathy, Lewy body diseases (e.g. Parkinson's disease), dementia, Huntington's disease and prion-based spongiform encephalopathy, and a combination thereof.

[0083J In certain embodiments, the present invention is directed to a compound of formula (X):

(CH2)p A/ \B
(X) wherein A and B are indolyl substituents; wherein A is substituted by A'(),) and B is substituted by Bi(y); wherein x and y are independently an integer from 0 to 4 and the sum of x and y is at least 1, provided that when x and y both equal 1, A' and B' are not both CO2H and are not both halogen;

pislor2;and each A1 and each B 1 are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, thio, thioether, cyano, nitro, halogen, carboxylic acid and sulfonic acid; or a pharmaceutically acceptable salt thereof.

[0084] In certain embodiments, the present invention is directed to a compound of formula (X):

(CH2)P
A/ \B
(X) wherein A and B are indolyl substituents; wherein A is substituted by A'(X) and B is substituted by B1C,); wherein x and y are independently an integer from 0 to 4 and the sum of x and y is at least 1;

p is l or 2; and each A1 and each B 1 are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, thio, thioether, cyano, nitro, and sulfonic acid;

or a pharmaceutically acceptable salt thereof.

[0085] In certain embodiments, the present invention is directed to a compound of formula (X):

A/ HZ)\B
(X) wherein A and B are indolyl substituents; wherein A is substituted by A'(),) and B is substituted by B1(y); wherein x and y are independently an integer from 0 to 4 and the sum of x and y is at least 1;

pis I or 2; and each A1 and each B I are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, thio, thioether, cyano, nitro, halogen, carboxylic acid and sulfonic acid; provided that the total number of CO2H substituents is not more than 1 and the total number of halogen substituents is not more than 1;

or a pharmaceutically acceptable salt thereof.

[0086] In certain embodiments, the present invention is directed to a compound of formula (X):

(CHZ)p A/ \B
(X) wherein A and B are indolyl substituents; wherein A is substituted by A l(,z) and B is substituted by B'C,); wherein x and y are independently an integer from 0 to 4 and the sum of x and y is at least 1;

p is 2; and each A 1 and each B1 are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, thio, thioether, cyano, nitro, halogen, carboxylic acid and sulfonic acid;

or a pharmaceutically acceptable salt thereof.

[0087] In certain embodiments of the present invention, the point of attachment for at least one of A and B is at the 1, 2 or 3 position of the indolyl.

[00881 In certain embodiments of the present invention, the point of attachment for both A and B is at the 1, 2 or 3 position of the indolyl.

[00891 In certain embodiments of the present invention, A is substituted by Ain at least one of the 4, 5, 6 or 7 positions of the indolyl.

[0090] In certain embodiments of the present invention, B is substituted by B'(y) in at least one of the 4, 5, 6 or 7 positions of the indolyl.

[0091] In certain embodiments of the present invention, the compound of formula (X) is:

A'(X) R'(y) \ / ~ I
I i N
Rz (XA) [0092] In certain embodiments of the present invention, the compound of formula (X) is:

A'(x) N bB N' (y) (XB) [0093] In certain embodiments of the present invention, x is I and A is at the position.

[0094] In certain embodiments of the present invention, y is I and B 1 is at the 6 position.

[0095] In certain embodiments of the present invention, y is I and B is at the position.

[0096] In certain embodiments of the present invention, A' is selected from the group consisting of halogen, OC1_3 alkyl, hydroxy and CO2H.

[0097] In certain embodiments of the present invention, Bl is selected from the group consisting of OCI_3 alkyl, hydroxy and CO2H.

[0098] In certain embodiments of the present invention, A' is selected from the group consisting of halogen and C02H.

[0099] In certain embodiments of the present invention, R' and Rz are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, arylalkyl, alkylcarbonyl, arylcarbonyl;
alkoxycarbonyl, aryloxycarbonyl, arylsulfonyl and alkylsulfonyl.

[00100] In certain embodiments of the present invention, B1 is selected from the group consisting of hydroxyl and CO2H.

[00101] In certain embodiments, the present invention is directed to a pharmaceutical composition comprising an effective amount of a compound of formula (X):

(CH2)p A/ \B
(X) wherein A and B are indolyl substituents; wherein A is substituted by Al(,t) and B is substituted by B1(Y); wherein x and y are independently an integer from 0 to 4;

p is l or 2; and each A1 and each Bl are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, hydroxyl, thio, thioether, cyano, nitro, halogen, carboxylic acid and sulfonic acid;

or a pharmaceutically acceptable salt thereof to treat a protein folding disorder, e.g., a neurodegenerative disease such as Alzheimer's disease, tauopathies, cerebral amyloid angiopathy, Lewy body diseases (e.g. Parkinson's disease), dementia, Huntington's disease and prion-based spongiform encephalopathy and a combination thereof.
1001021 In certain embodiments, the invention is directed to a method of treating a protein folding disorder comprising administering to a patient in need thereof an effective amount of a compound which attenuates the increase in thioflavin T fluorescence by greater than 30%; greater than 60%; or greater than 90%;
relative to beta-amyloid with vehicle as a control, at 20 hours when subjected to a beta-amyloid thioflavin T aggregation assay. In certain such embodiments, the compound is a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X).
[001031 In certain embodiments, the invention is directed to a method of treating a protein folding disorder comprising administering to a patient in need thereof an effective amount of a compound which attenuates the increase in thioflavin T fluorescence by greater than 30%; greater than 60%; or greater than 90%;
relative to beta-amyloid with vehicle as a control, at 30 hours when subjected to a beta-amyloid thioflavin T aggregation assay. In certain such embodiments, the compound is a compound of formula (I), (11), (111), (IV), (V), (VI), (VII), (VIII), (IX) or (X).

[00104] In certain embodiments, the invention is directed to a method of treating a protein folding disorder comprising administering to a patient in need thereof an effective amount of a compound which attenuates the increase in thioflavin S (ThS) fluorescence by greater than 30%; greater than 60%; or greater than 90%;
relative to tau with vehicle as a control, at 30 hours when subjected to a tau thioflavin S (ThS) aggregation assay. In certain such embodiments, the compound is a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X).

[00105] In certain embodiments, the invention is directed to a method of treating a protein folding disorder comprising administering to a patient in need thereof an effective amount of a compound which attenuates the increase in thioflavin T (ThT) fluorescence by greater than 30%; greater than 60%; or greater than 90%;
relative to alpha-synuclein with vehicle as a control, at 30 hours when subjected to an alpha-synuclein thioflavin T (ThT) aggregation assay. In certain such embodiments, the compound is a compound of formula (I), (11), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X).

[00106] In certain embodiments, the invention is directed to a method of treating a protein folding disorder comprising administering to a subject in need thereof an effective amount of a compound which, when co-incubated with beta-amyloid, causes the peptide to exhibit circular dichroism, at 193nm after 48 hours, of less than that of beta amyloid with vehicle. In certain such embodiments, the compound is a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X).

[00107] In certain embodiments, the invention is directed to a method of treating a protein folding disorder comprising administering to a subject in need thereof an effective amount of a compound which, when co-incubated with beta-amyloid, causes the peptide to exhibit circular dichroism, at 193nm after 48 hours, of at least 2 mdeg less than that of beta amyloid with vehicle. In certain such embodiments, the compound is a compound of formula ((I), (11), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X).

[00108] In certain embodiments, the invention is directed to a method of treating a protein folding disorder comprising administering to a subject in need thereof an effective amount of a compound which, when co-incubated with beta-amyloid, causes the peptide to exhibit circular dichroism, at 193nm after 72 hours, of less than that of beta amyloid with vehicle. In certain such embodiments, the compound is a compound of formula (1), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X).

[00109] In certain embodiments, the invention is directed to a method of treating a protein folding disorder comprising administering to a subject in need thereof an effective amount of a compound which, when co-incubated with beta-amyloid, causes the peptide to exhibit circular dichroism, at 193nm after 72 hours, of at least 2 mdeg less than that of beta amyloid with vehicle. In certain such embodiments, the compound is a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X).

[00110] In certain embodiments, the invention is directed to a method of treating a protein folding disorder comprising administering to a subject in need thereof an effective amount of a compound which, when co-incubated with beta-amyloid, causes the peptide to exhibit circular dichroism, at 193nm after 72 hours, of at least 5 mdeg less than that of beta amyloid with vehicle. In certain such embodiments, the compound is a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X).

[00111] In certain embodiments, the invention is directed to a method of treating a protein folding disorder comprising administering to a patient in need thereof, a compound whose aromatic or heteroaromatic substituents each exhibit sufficient gas-phase cation-7t binding energy to cationic residues of the protein to treat the protein folding disorder. In certain such embodiments, the compound is a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X).
[00112] In certain embodiments, the invention is directed to a method of treating a protein folding disorder comprising administering to a patient in need thereof, a compound whose aromatic or heteroaromatic substituents each exhibit a gas-phase cation-n binding energy to cationic residues of the protein of at least 15 kcal/mol in a RHF/6-31G(d)//RHF/3-21G optimization calculation, as implemented within the Gaussian98 computer program (Rev. A.9. 1998, Gaussian Inc., Pittsburgh, PA, U.S.A., See Example 20). In certain such embodiments, the compound is a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X).

[00113] In certain embodiments, the invention is directed to a method for inhibiting tau protein aggregation or for treating a protein folding disorder comprising administering a compound which attenuates the increase in thioflavin S
fluorescence by greater than 30% at 20 hours, relative to tau441 with vehicle as a control, in a tau aggregation assay. In certain such embodiments, the compound is a compound of formula (I), (11), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X).

[00114] In certain embodiments, the invention is directed to a method for inhibiting tau protein aggregation or for treating a protein folding disorder comprising administering a compound which attenuates an increase in thioflavin S
fluorescence by greater than 60% at 20 hours, relative to tau441 with vehicle as a control, in a tau aggregation assay. In certain such embodiments, the compound is a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X).

[001151 In certain embodiments, the invention is directed to a method for inhibiting tau protein aggregation or for treating a protein folding disorder comprising administering a compound which attenuates an increase in thioflavin S
fluorescence by greater than 90% at 20 hours, relative to tau441 with vehicle as a control, in a tau aggregation assay. In certain such embodiments, the compound is a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X).

[00116] In certain embodiments, the invention is directed to a method for inhibiting tau protein aggregation or for treating a protein folding disorder comprising administering a compound which attenuates an increase in thioflavin S
fluorescence by greater than 30% at 30 hours, relative to tau441 with vehicle as a control, in a tau aggregation assay. In certain such embodiments, the compound is a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X).

[001171 In certain embodiments, the invention is directed to a method for inhibiting tau protein aggregation or for treating a protein folding disorder comprising administering a compound which attenuates an increase in thioflavin S
fluorescence by greater than 60% at 30 hours, relative to tau441 with vehicle as a control, in a tau aggregation assay. In certain such embodiments, the compound is a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X).

[00118] In certain embodiments, the invention is directed to a method for inhibiting tau protein aggregation or for treating a protein folding disorder comprising administering a compound which attenuates an increase in thioflavin S
fluorescence by greater than 90% at 30 hours, relative to tau441 with vehicle as a control, in a tau aggregation assay. In certain such embodiments, the compound is a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X).

[00119] In certain embodiments, the invention is directed to a method of treating a protein folding disorder comprising administering to a patient in need thereof an effective amount of a compound which:

(i) attenuates the increase in thioflavin T fluorescence by greater than 30%, relative to beta-amyloid with vehicle as a control, at 20 hours when subjected to a beta-amyloid thioflavin T aggregation assay, and (ii) attenuates an increase in fluorescence by greater than 30%, greater than 60% or greater than 90% at 20 hours, relative to tau441 with vehicle as a control, in a tau thioflavin S aggregation assay.

[001201 In certain embodiments, the invention is directed to a method of treating a protein folding disorder comprising administering to a patient in need thereof an effective amount of a compound which:

(i) attenuates the increase in thioflavin T fluorescence by greater than 60%, relative to beta-amyloid with vehicle as a control, at 20 hours when subjected to a beta-amyloid thioflavin T aggregation assay, and (ii) attenuates an increase in fluorescence by greater than 30%, greater than 60% or greater than 90% at 20 hours, relative to tau44l with vehicle as a control, in a tau thioflavin S aggregation assay.

[00121] In certain embodiments, the invention is directed to a method of treating a protein folding disorder comprising administering to a patient in need thereof an effective amount of a compound which:

(i) attenuates the increase in thioflavin T fluorescence by greater than 90%, relative to beta-amyloid with vehicle as a control, at 20 hours when subjected to a beta-amyloid thioflavin T aggregation assay, and (ii) attenuates an increase in fluorescence by greater than 30%, greater than 60% or greater than 90% at 20 hours, relative to tau441 with vehicle as a control, in a tau thioflavin S aggregation assay.

[00122] In certain embodiments, the invention is directed to a method of treating a protein folding disorder comprising administering to a patient in need thereof an effective amount of a compound which:

(i) attenuates the increase in thioflavin T fluorescence by greater than 30%, relative to beta-amyloid with vehicle as a control, at 30 hours when subjected to a beta-amyloid thioflavin T aggregation assay, and (ii) attenuates an increase in fluorescence by greater than 30%, greater than 60% or greater than 90% at 30 hours, relative to tau441 with vehicle as a control, in a tau thioflavin S aggregation assay.

[001231 In certain embodiments, the invention is directed to a method of treating a protein folding disorder comprising administering to a patient in need thereof an effective amount of a compound which:

(i) attenuates the increase in thioflavin T fluorescence by greater than 60%, relative to beta-amyloid with vehicle as a control, at 30 hours when subjected to a beta-amyloid thioflavin T aggregation assay, and (ii) attenuates an increase in fluorescence by greater than 30%, greater than 60% or greater than 90% at 30 hours, relative to tau441 with vehicle as a control, in a tau thioflavin S aggregation assay.

[00124} In certain embodiments, the invention is directed to a method of treating a protein folding disorder comprising administering to a patient in need thereof an effective amount of a compound which:

(i) attenuates the increase in thioflavin T fluorescence by greater than 90%, relative to beta-amyloid with vehicle as a control, at 30 hours when subjected to a beta-amyloid thioflavin T aggregation assay, and (ii) attenuates an increase in fluorescence by greater than 30%, greater than 60% or greater than 90% at 30 hours, relative to tau441 with vehicle as a control, in a tau thioflavin S aggregation assay.

[001251 In certain embodiments, the invention is directed to a compound which attenuates an increase in thioflavin S fluorescence by greater than 30% at 20 hours, relative to tau441 with vehicle as a control, in a tau aggregation assay.

[00126J In certain embodiments, the invention is directed to compound which attenuates an increase in thioflavin S fluorescence by greater than 60% at 20 hours, relative to tau441 with vehicle as a control, in a tau aggregation assay.

[00127] In certain embodiments, the invention is directed to a compound which attenuates an increase in thioflavin S fluorescence by greater than 90% at 20 hours, relative to tau441 with vehicle as a control, in a tau aggregation assay.

[00128] In certain embodiments, the invention is directed to a compound which attenuates an increase in thioflavin S fluorescence by greater than 30% at 30 hours, relative to tau441 with vehicle as a control, in a tau aggregation assay.

[00129] In certain embodiments, the invention is directed to a compound which attenuates an increase in thioflavin S fluorescence by greater than 60% at 30 hours, relative to tau441 with vehicle as a control, in a tau aggregation assay.

[00130] In certain embodiments, the invention is directed to a compound which attenuates an increase in thioflavin S fluorescence by greater than 90% at 30 hours, relative to tau441 with vehicle as a control, in a tau aggregation assay.

[00131] In certain embodiments, the invention is directed to a compound which:

(i) attenuates the increase in thioflavin T fluorescence by greater than 30%, relative to beta-amyloid with vehicle as a control, at 20 hours when subjected to a beta-amyloid thioflavin T aggregation assay, and (ii) attenuates an increase in fluorescence by greater than 30%, greater than 60% or greater than 90% at 20 hours, relative to tau441 with vehicle as a control, in a tau thioflavin S aggregation assay.

[00132] In certain embodiments, the invention is directed to a compound which:

(i) attenuates the increase in thioflavin T fluorescence by greater than 60%, relative to beta-amyloid with vehicle as a control, at 20 hours when subjected to a beta-amyloid thioflavin T aggregation assay, and (ii) attenuates an increase in fluorescence by greater than 30%, greater than 60% or greater than 90% at 20 hours, relative to tau441 with vehicle as a control, in a tau thioflavin S aggregation assay.

[00133] In certain embodiments, the invention is directed to a compound which:

(i) attenuates the increase in thioflavin T fluorescence by greater than 90%, relative to beta-amyloid with vehicle as a control, at 20 hours when subjected to a beta-amyloid thioflavin T aggregation assay, and (ii) attenuates an increase in fluorescence by greater than 30%, greater than 60% or greater than 90% at 20 hours, relative to tau441 with vehicle as a control, in a tau thioflavin S aggregation assay.

[00134] In certain embodiments, the invention is directed to a compound which:

(i) attenuates the increase in thioflavin T fluorescence by greater than 30%, relative to beta-amyloid with vehicle as a control, at 30 hours when subjected to a beta-amyloid thioflavin T aggregation assay, and (ii) attenuates an increase in fluorescence by greater than 30%, greater than 60% or greater than 90% at 30 hours, relative to tau441 with vehicle as a control, in a tau thioflavin S aggregation assay.

[00135] In certain embodiments, the invention is directed to a compound which:

(i) attenuates the increase in thioflavin T fluorescence by greater than 60%, relative to beta-amyloid with vehicle as a control, at 30 hours when subjected to a beta-amyloid thioflavin T aggregation assay, and (ii) attenuates an increase in fluorescence by greater than 30%, greater than 60% or greater than 90% at 30 hours, relative to tau441 with vehicle as a control, in a tau thioflavin S aggregation assay.

[00136] In certain embodiments, the invention is directed to a coinpound which:

(i) attenuates the increase in thioflavin T fluorescence by greater than 90%, relative to beta-amyloid with vehicle as a control, at 30 hours when subjected to a beta-amyloid thioflavin T aggregation assay, and (ii) attenuates an increase in fluorescence by greater than 30%, greater than 60% or greater than 90% at 30 hours, relative to tau441 with vehicle as a control, in a tau thioflavin S aggregation assay.

[00137] In certain embodiments disclosed herein, the aggregation assay utilizes the conditions set forth in Figure 10 (A-D).

[00138] In certain embodiments, the invention is directed to a method for the treatment of a protein folding disorder in a subject comprising administering an effective amount of a therapeutic agent to said patient, wherein said therapeutic agent binds to at least one of a BXBB, BBXB, AXBBXB or BXBBXA receptor site of a protein associated with the protein folding disorder. The binding of therapeutic agents at the BXBB, BBXB, AXBBXB and BXBBXA receptor sites is described in FEBS Letters 2005, "The 'promiscuous drug concept' with applications to Alzheimer's disease", Stephenson VC et al. 2005. FEBS Lett 579:1338-42, the disclosure of which is hereby incorporated by reference. The FEBS Letters discusses that, arguably, Alzheimer's disease (AD) is a multifactorial syndrome, rather than single disease, arising from a complex array of neurocheinical factors. Numerous studies on the molecular pathogenesis of AD implicate a diversity of factors ranging from RECTIFIED SHEET (RULE 91) neurotoxic peptides (0-amyloid) to inflammatory processes (interleukins), but all culminating in a common neuropathology. This diversity of molecular causation is an impediment to the design of effective therapies for AD. To address this design problem, we sought to identify a single, common motif (a "common receptor") shared by multiple structurally and functionally diverse proteins implicated in AD.
This search revealed the presence of a common BBXB peptide motif and upon refinement, an AXBBXB motif; these regions can be exploited for the design of a "promiscuous drug" that exploits a "one-drug-multiple-receptors" therapeutic strategy for AD.
[00139] The novel concept of a promiscuous drug addresses the emerging need for one-drug-multiple-target therapeutics. A promiscuous drug candidate is not a collection of different drug molecules combined in a single pill to act on a multitude of receptors implicated in the pathogenesis of a single disease; rather, it is a single entity that occupies specific and discrete volumes of biological space common to multiple different receptor targets. Given its complex multifactorial etiology, it is highly probable that AD and other protein folding disorders may benefit from such a promiscuous drug strategy. The BBXB and AXBBXB motifs identified in this study represent targets worthy of promiscuous drug design.

[00140] In addition to providing an enabling strategy for promiscuous drug design, this unbiased computer-driven identification of the BBXB/AXBBXB motifs may also provide fundamental insights into the biochemical basis of AD. The effective binding of the glycosaminoglycan (GAG), heparin, to the identified domains in 27 Alzheimer's associated proteins also strengthens the design possibilities for promiscuous drug therapeutics, as well as providing a harmonized structural basis for understanding and combating the immunopathology of AD.

[00141] In certain embodiments, the therapeutic agent has a binding energy to the lA1vIB PDB Structure of the HHQK region of A(3 greater than -54.4 kcal/mol at the Hisl3-Hisl4 region.

[00142] In certain embodiments, the therapeutic agent has a binding energy to the lAMB PDB Structure of the HHQK region of A(3 greater than -60.0 kcal/mol at the His13-His14 region.

[00143] In certain embodiments, the therapeutic agent has a binding energy to the IAMB PDB Structure of the HHQK region of Af3 greater than -65.0 kcal/mol at the His13-His14 region.

[00144] In certain embodiments, the therapeutic agent has a binding energy to the IAMB PDB Structure of the HHQK region of A(3 greater than -70.0 kcal/mol at the Hisl3-Hisl4 region.

[00145] In certain embodiments, the therapeutic agent has a binding energy to the I AMC PDB Structure of the HHQK region of A(3 greater than -44.6 kcaUmol at the His13-His14 region.

[00146] In certain embodiments, the therapeutic agent has a binding energy to the IAMC PDB Structure of the HHQK region of A(3 greater than -50.0 kcal/mol at the Hisl3-His14 region.

[00147] In certain embodiments, the therapeutic agent has a binding energy to the IAMC PDB Structure of the HHQK region of A[I greater than -55.0 kcal/mol at the His13-His14 region.

[00148] In certain embodiments, the therapeutic agent has a binding energy to the l AMC PDB Structure of the HHQK region of A(3 greater than -60.0 kcal/mol at the His 13-His 14 region.

[00149] In certain embodiments, the therapeutic agent has a binding energy to the l AML PDB Structure of the HHQK region of A(3 greater than -35.6 kcal/mol at the His13-His14 region.

[00150] In certain embodiments, the therapeutic agent has a binding energy to the 1AML PDB Structure of the HHQK region of A(3 greater than -40.0 kcal/mol at the His13-Hisl4 region.

[001511 In certain embodiments, the therapeutic agent has a binding energy to the 1AML PDB Structure of the HHQK region of AQ greater than -45.0 kcaUmol at the His13-His14 region.

[00152] In certain embodiments, the therapeutic agent has a binding energy to the 1 AML PDB Structure of the HHQK region of A(3 greater than -50.0 kcal/mol at the Hisl3-Hisl4 region.

[00153] In certain embodiments, the therapeutic agent has a binding energy to the 1 BA4 PDB Structure of the HHQK region of Ap greater than -27.1 kcal/mol at the His13-His14 region.

[00154] In certain embodiments, the therapeutic agent has a binding energy to the 1BA4 PDB Structure of the HHQK region ofA[i greater than -30.0 kcal/mol at the His13-His14 region.

[00155] In certain embodiments, the therapeutic agent has a binding energy to the IBA4 PDB Structure of the HHQK region of A(3 greater than -35.0 kcaUmol at the His13-His14 region.

[00156] In certain embodiments, the therapeutic agent has a binding energy to the 1BA4 PDB Structure of the HHQK region of Ap greater than -40.0 kcal/mol at the Hisl3-Hisl4 region.

[00157] In certain embodiments, the therapeutic agent has a binding energy to the 1IYT PDB Structure of the HHQK region of A(3 greater than -36.8 kcal/mol at the His13-His14 region.

[00158] In certain embodiments, the therapeutic agent has a binding energy to the 1IYT PDB Structure of the HHQK region of A[3 greater than -40.0 kcal/mol at the Hisl3-Hisl4 region.

100159] In certain embodiments, the therapeutic agent has a binding energy to the 1IYT PDB Structure of the HHQK region of A[i greater than -45.0 kcal/mol at the His13-His14 region.

[00160] In certain embodiments, the therapeutic agent has a binding energy to the 1IYT PDB Structure of the HHQK region of A(3 greater than -50.0 kcal/mol at the His13-His14 region.

[00161] In certain embodiments, the therapeutic agent has a binding energy to the 2BP4 PDB Structure of the HHQK region of A(3 greater than -32.7 kcal/mol at the His13-His14 region.

[00162] In certain embodiments, the therapeutic agent has a binding energy to the 2BP4 PDB Structure of the HHQK region of AR greater than -35.0 kcal/mol at the His13-His14 region.

[00163] In certain embodiments, the therapeutic agent has a binding energy to the 2BP4 PDB Structure of the HHQK region of A(i greater than -40.0 kcal/mol at the His13-His14 region.

[00164] In certain embodiments, the therapeutic agent has a binding energy to the 2BP4 PDB Structure of the HHQK region of AR greater than -40.0 kcal/mol at the His13-His14 region.

[001651 In certain embodiments, the therapeutic agent has a binding energy to the IAMB PDB Structure of the HHQK region of A(3 greater than -43.5 kcaUmol at the His ] 3-Lys 16 region.

[00166] In certain embodiments, the therapeutic agent has a binding energy to the IAMB PDB Structure of the HHQK region of A(3 greater than -45.0 kcaUmol at the His13-Lys16 region.

[00167] In certain embodiments, the therapeutic agent has a binding energy to the IAMB PDB Structure of the HHQK region of A(3 greater than -50.0 kcal/mol at the His13-Lys16 region.

[00168] In certain embodiments, the therapeutic agent has a binding energy to the IAMB PDB Structure of the HHQK region of A(3 greater than -55.0 kcaUmol at the His13-Lys16 region.

[00169] In certain embodiments, the therapeutic agent has a binding energy to the 1AMC PDB Structure of the HHQK region of A[3 greater than -34.3 kcal/mol at the Hisl3-Lysl6 region.

[00170) In certain embodiments, the therapeutic agent has a binding energy to the I AMC PDB Structure of the HHQK region of A(3 greater than -40.0 kcaUmol at the His13-Lys16 region.

[00171] In certain embodiments, the therapeutic agent has a binding energy to the 1 AMC PDB Structure of the HHQK region of A[i greater than -45.0 kcallmol at the His 13-Lys 16 region.

[00172] In certain embodiments, the therapeutic agent has a binding energy to the 1AMC PDB Structure of the HHQK region of A(3 greater than -50.0 kcal/mol at the His13-Lys16 region.

[00173] In certain embodiments, the therapeutic agent has a binding energy to the lAML PDB Structure of the HHQK region of A[3 greater than -22.3 kcal/mol at the His13-LysI6 region.

1001741 In certain embodiments, the therapeutic agent has a binding energy to the lAML PDB Structure of the HHQK region of A[3 greater than -25.0 kcaUmol at the His 13-Lys 16 region.

[00175] In certain embodiments, the therapeutic agent has a binding energy to the l AML PDB Structure of the HHQK region of A(3 greater than -30.0 kcal/mol at the His13-Lys16 region.

[00176] In certain embodiments, the therapeutic agent has a binding energy to the IAML PDB Structure of the HHQK region of A(3 greater than -35.0 kcal/mol at the His 13-Lys 16 region.

[00177] In certain embodiments, the therapeutic agent has a binding energy to the 1 BA4 PDB Structure of the HHQK region of A(3 greater than -46.0 kcai/mol at the Hisl3-Lysl6 region.

[00178] In certain embodiments, the therapeutic agent has a binding energy to the 1BA4 PDB Structure of the HHQK region of A[i greater than -50.0 kcaUmol at the Hisl3-Lysl6 region.

[00179] In certain embodiments, the therapeutic agent has a binding energy to the 1 BA4 PDB Structure of the HHQK region of A(3 greater than -55.0 kcal/mol at the Hisl3-Lysl6 region.

[00180] In certain embodiments, the therapeutic agent has a binding energy to the 1BA4 PDB Structure of the HHQK region of A[3 greater than -60.0 kcal/mol at the His13-Lys16 region.

[00181] In certain embodiments, the therapeutic agent has a binding energy to the 1IYT PDB Structure of the HHQK region of A(3 greater than -17.3 kcal/mol at the His13-Lys16 region.

[00182] In certain embodiments, the therapeutic agent has a binding energy to the 1IYT PDB Structure of the HHQK region of A(3 greater than -20.0 kcal/mol at the His13-Lys16 region.

[00183] In certain embodiments, the therapeutic agent has a binding energy to the 1IYT PDB Structure of the HHQK region of A(3 greater than -25.0 kcal/mol at the His13-Lys16 region.

1001841 In certain embodiments, the therapeutic agent has a binding energy to the 1IYT PDB Structure of the HHQK region of Aa greater than -30.0 kcal/mol at the His13-Lys16 region.

[00185] In certain embodiments, the therapeutic agent has a binding energy to the 2BP4 PDB Structure of the HHQK region of A[i greater than -48.6 kcaUmol at the His13-Lys16 region.

[00186] In certain embodiments, the therapeutic agent has a binding energy to the 2BP4 PDB Structure of the HHQK region of A[3 greater than -50.0 kcal/mol at the His13-Lys16 region.

[00187] In certain embodiments, the therapeutic agent has a binding energy to the 2BP4 PDB Structure of the HHQK region of AR greater than -55.0 kcal/mol at the His13-Lys16 region.

[00188] In certain embodiments, the therapeutic agent has a binding energy to the 2BP4 PDB Structure of the HHQK region of A[i greater than -60.0 kcal/mol at the His13-Lys16 region.

1001891 For purposes of this invention, the term "binding energy" means the energy required to separate particles from a molecule or atom or nucleus.
Therefore, the more negative the number is, the more energy is required for separation, thus a greater binding energy, and thus greater binding affinity.

[00190] In certain embodiments, the HHQK region includes the portions described above and in Table 18A of Example 18.

[00191] In certain embodiments, the therapeutic agent has a binding energy to the HHQK region of A[3 that is 2% greater than the binding energy of L-tryptophan to the HHQK region of A13.

[00192] In certain embodiments, the therapeutic agent has a binding energy to the HHQK region of A(3 that is 5% greater than the binding energy of L-tryptophan to the HHQK region of AB.

[00193] In certain embodiments, the therapeutic agent has a binding energy to the HHQK region of A[I that is 10% greater than the binding energy of L-tryptophan to the HHQK region of AB.

[00194] In certain embodiments, the binding energy is measured using the CHARMM27 force field and explicit solvation.

[00195] In other embodiments of the invention, the therapeutic agent is a compound as disclosed herein. In other embodiments, the therapeutic agent is not L-tryptophan. In other embodiments, the therapeutic agent is not a tryptophan dipeptide.

[00196] In certain embodiments of the invention, the protein folding disorder is Alzheimer's Disease.

[00197] In certain embodiments of the invention, the therapeutic agent has a bonding distance to the BXBB, BBXB, AXBBXB or BXBBXA receptor site of from about 1.63 to about 3.48 A.

[00198] In certain embodiments of the disclosed method, the protein folding disorder being treated is a neurodegenerative disease.

[00199] In certain embodiments of the disclosed method, the neurodegenerative disease is selected from the group consisting of tauopathies, cerebral amyloid angiopathy, Lewy body diseases (e.g. Parkinson's disease), Alzheimer's disease, dementia, Huntington's disease, prion-based spongiform encephalopathy and a combination thereof.

[00200] In certain embodiments of the disclosed method, the neurodegenerative disease is Alzheimer's disease.

[00201] In certain embodiments, the present invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an effective amount of a compound of formula (I), (II), (III), (IV), (V), (VI), (VII) or (VIII) to treat a protein folding disorder, e.g., a neurodegenerative disease such as, tauopathies, cerebral amyloid angiopathy, Lewy body diseases (e.g. Parkinson's disease), Alzheimer's disease, dementia, Huntington's disease, prion-based spongiform encephalopathy and a combination thereof.

[00202] In certain embodiments, the present invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an effective amount of a compound of formula (I), (II), (111), (IV), (V), (VI), (VII) or (VIII) to treat systemic amyloidoses, particularly those affecting the peripheral nerves, spleen and pancreas.

[00203] In certain embodiments, the invention is directed to a method for treating a protein folding disorder comprising administering a compound or pharmaceutical composition as disclosed herein to a subject wherein the subject is treated for the protein folding disorder.

[00204] In certain embodiments, the invention is directed to a method for treating a protein folding disorder comprising administering an effective amount of a compound or pharmaceutical composition as disclosed herein to a patient in need thereof.

[00205] In certain embodiments, the compounds of the present invention are non-peptides.

1002061 As used herein, the term "alkyl" means a linear or branched saturated aliphatic hydrocarbon group having a single radical and 1-10 carbon atoms.
Examples of alkyl groups include methyl, propyl, isopropyl, butyl, n-butyl, isobutyl, sec-butyl, tert-butyl, and pentyl. A branched alkyl means that one or more alkyl groups such as, e.g., methyl, ethyl or propyl, replace one or both hydrogens in a-CHz-group of a linear alkyl chain. The term "lower alkyl" means an alkyl of 1-3 carbon atoms.

[00207] The term "alkoxy" means an "alkyl" as defined above connected to an oxygen radical.

[002081 The term "cycloalkyl" means a non-aromatic mono- or multicyclic hydrocarbon ring system having a single radical and 3-12 carbon atoms.
Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclopentyl, and cyclohexyl.
Exemplary multicyclic cycloalkyl rings include adamantyl and norbornyl.

[00209] The term "alkenyl" means a linear or branched aliphatic hydrocarbon group containing a carbon-carbon double bond having a single radical and 2-10 carbon atoms.

[00210] A "branched" alkenyl means that one or more alkyl groups such as, e.g., methyl, ethyl or propyl replace one or both hydrogens in a -CH2- or -CH=
linear alkenyl chain. Exemplary alkenyl groups include ethenyl, 1- and 2-propenyl, 1-, 2-and 3-butenyl, 3-methylbut-2-enyl, heptenyl, octenyl and decenyl.

[00211] The term "cycloalkenyl" means a non-aromatic monocyclic or multicyclic hydrocarbon ring system containing a carbon-carbon double bond having a single radical and 3 to 12 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopropenyl, cyclopentenyl, cyclohexenyl or cycloheptenyl. An exemplary multicyclic cycloalkenyl ring is norbornenyl.

[00212] The term "alkynyl" means a linear or branched aliphatic hydrocarbon group containing a carbon-carbon triple bond having a single radical and 2-10 carbon atoms.

[00213] A "branched" alkynyl means that one or more alkyl groups such as, e.g., methyl, ethyl or propyl replace one or both hydrogens in a -CH2- linear alkynyl chain.

[00214] The term "cycloalkynyl" means a non-aromatic monocyclic or multicyclic hydrocarbon ring system containing a carbon-carbon triple bond having a single radical and 3 to 12 carbon atoms.

[00215] The term "aryl" means a carbocyclic aromatic ring system containing one, two or three rings which may be attached together in a pendent manner or fused, and containing a single radical. Exemplary aryl groups include phenyl, naphthyl and acenaphthyl.

[002161 The term "heteroaryl" means unsaturated heterocyclic radicals.

Exemplary heteroaryl groups include unsaturated 3 to 6 membered hetero-monocyclic groups containing 1 to 4 nitrogen atoms, such as, e.g., pyrrolyl, pyridyl, pyrimidyl, and pyrazinyl; unsaturated condensed heterocyclic groups containing I to 5 nitrogen atoms, such as, e.g., indolyl, quinolyl and isoquinolyl; unsaturated 3 to 6-membered hetero-monocyclic groups containing an oxygen atom, such as, e.g., furyl;
unsaturated 3 to 6 membered hetero-monocyclic groups containing a sulfur atom, such as, e.g., thienyl; unsaturated 3 to 6 membered hetero-monocyclic groups containing 1 to oxygen atoms and 1 to 3 nitrogen atoms, such as, e.g., oxazolyl; unsaturated condensed heterocyclic groups containing 1 to 2 oxygen atoms and I to 3 nitrogen atoms, such as, e.g., benzoxazolyl; unsaturated 3 to 6 membered hetero-monocyclic groups containing I to 2 sulfur atoms and I to 3 nitrogen atoms, such as, e.g., thiazolyl; and unsaturated condensed heterocyclic group containing I to 2 sulfur atoms and 1 to 3 nitrogen atoms, such as, e.g., benzothiazolyl. The term "heteroaryl"
also includes unsaturated heterocyclic radicals, wherein "heterocyclic" is as previously described, in which the heterocyclic group is fused with an aryl group, in which aryl is as previously described. Exemplary fused radicals include benzofuran, benzodioxole and benzothiophene.

[00217] The term "carbonyl", whether used alone or with other terms, such as, e.g., "alkoxycarbonyl", is (C=O).

[00218] The term "alkylcarbonyl" includes radicals having alkyl radicals, as defined above, attached to a carbonyl radical.

[00219] The term "carboxylic acid" is CO2H.

[00220] All of the cyclic ring structures disclosed herein can be attached at any point where such connection is possible, as recognized by one skilled in the art.

[00221] The terms "bi-indole" and "bis-indole" are used interchangeably.

[00222] As used herein, the term "subject" includes a human or an animal such as, e.g., a companion animal or livestock.

[00223] The term "patient" includes a subject in need of therapeutic treatment.
[00224] As used herein, the tenn "halogen" or "halo" includes fluoride, bromide, chloride, iodide or astatide.

[00225] For purposes of the present invention the abbreviation "Trp" means tryptophan.

[00226] There are many different isoforms of tau which can be utilized in a tau aggregation assay. The particular tau isoform utilized in the present invention is not meant to limit the scope of the invention which encompasses tau aggregation assays utilizing any suitable tau isomer.

[00227] For purposes of the present invention, wherein the formula includes a q, q, and/or a q2 variable, when the q, q, and/or a qZ variable is less than 4, it is understood that the base structure will include hydrogen substituents where necessary (e.g., on the aromatic ring) to complete valence.

[00228] The invention disclosed herein is meant to encompass all pharmaceutically acceptable salts thereof of the disclosed compounds. The pharmaceutically acceptable salts include, but are not limited to, metal salts such as, e.g., sodium salt, potassium salt, cesium salt and the like; alkaline earth metals such as, e.g., calcium salt, magnesium salt and the like; organic amine salts such as, e.g., triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt and the like;
inorganic acid salts such as, e.g., hydrochloride, hydrobromide, sulfate, phosphate and the like;
organic acid salts such as, e.g., formate, acetate, trifluoroacetate, maleate, fumarate, tartrate and the like; sulfonates such as, e.g., methanesulfonate, benzenesulfonate, p-toluenesulfonate, and the like; amino acid salts such as, e.g., arginate, asparginate, glutamate and the like.

(002291 The invention disclosed herein is also meant to encompass all prodrugs of the disclosed compounds. Prodrugs are considered to be any covalently bonded carriers which release the active parent drug in vivo. An example of a prodrug would be an ester which is processed in vivo to a carboxylic acid or salt thereof.

[00230] The invention disclosed herein is also meant to encompass the in vivo metabolic products of the disclosed compounds. Such products may result for example from the oxidation, reduction, hydrolysis, amidation, esterification and the like of the administered compound, primarily due to enzymatic processes.
Accordingly, the invention includes compounds produced by a process comprising contacting a compound of this invention with a mammal for a period of time sufficient to yield a metabolic product thereof. Such products typically are identified by preparing a radiolabelled compound of the invention, administering it parenterally in a detectable dose to an animal such as, e.g., a rat, mouse, guinea pig, monkey, or to man, allowing sufficient time for metabolism to occur and isolating its conversion products from the urine, blood or other biological samples. One skilled in the art recognizes that interspecies pharmacokinetic scaling can be used to study the underlining similarities (and differences) in drug disposition among species, to predict drug disposition in an untested species, to define pharmacokinetic equivalence in various species, and to design dosage regimens for experimental animal models, as discussed in Mordenti, Man versus Beast: Pharmacokinetic Scaling in Mammals, 1028, Journal of Pharmaceutical Sciences, Vol. 75, No. 11, November 1986.

[00231] The invention disclosed herein is also meant to encompass the disclosed compounds being isotopically-labelled by having one or more atoms replaced by an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as, e.g., 2H~ 3H, isC, 14C, 15N, 180, t7o, siP, 32P, ssS, "F, and 36C1, respectively.
Some of the compounds disclosed herein may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms.
The present invention is also meant to encompass all such possible forms as well as their racemic and resolved forms and mixtures thereof. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended to include both E and Z geometric isomers.
All tautomers are intended to be encompassed by the present invention as well.
[00232] As used herein, the term "stereoisomers" is a general term for all isomers of individual molecules that differ only in the orientation of their atoms in space. It includes enantiomers and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereomers).

[00233] The term "chiral center" refers to a carbon atom to which four different groups are attached.

[002341 The term "enantiomer" or "enantiomeric" refers to a molecule that is nonsuperimposeable on its mirror image and hence optically active wherein the enantiomer rotates the plane of polarized light in one direction and its mirror image rotates the plane of polarized light in the opposite direction.

[00235] The term "racemic" refers to a mixture of equal parts of enantiomers and which is optically inactive.

[00236] The term "resolution" refers to the separation or concentration or depletion of one of the two enantiomeric forms of a molecule.
BRIEF DESCRIPTION OF THE DRAWINGS
[00237] Figure 1(A-G) depicts the inhibition of A(31-40 aggregation bycompounds of the present invention as shown in Example 8, as measured by Thioflavin T (ThT) fluorescence.

[00238] Figure 2 depicts the dose-response effect of a compound of the present invention on A[31"40 aggegation in kinetic ThT assay as shown in Example 8.

[00239] Figure 3 depicts dose-response curves for inhibition ofA[3'-40 aggregation by a compound of the present invention and a control as shown in Example 8.

[00240] Figure 4 depicts the inhibition of A[3'40 aggregation by a compound of the present invention as shown in Example 8 in a "seeded" ThT assay.

[00241] Figure 5 depicts disaggregation of A[3l-40 by a compound of the present invention and a control in a ThT assay as shown in Example 8.

[00242] Figures 6A-C depict circular dichroism studies of compounds of the present invention and a control as shown in Example 8.

[00243] Figure 7A-C depicts the inhibition of A(31-42 aggregation by compounds of the current invention as shown in Example 8, as measured by ThT
fluorescence.

[00244] Figure 8 depicts the dose-response curves for inhibition of A[3'4Z
aggregation by compounds of the current invention as shown in Example 8, as measured by ThT fluorescence.

[00105] Figure 9 depicts an 1H NMR binding study for a compound of the current invention to A(31-40, as shown in Example 8.

[00106] Figure 10A-E depicts the inhibition, or lack thereof, of tau aggregation,' seen as both a reduced rate of tau aggregation and reduced equilibrium or plateau level of aggregation, by compounds of the current invention and nicotinic acid, as shown in Example 8, as measured by Thioflavin S (ThS) fluorescence. The inhibition of tau aggregation is by synthesized bi-aromatic compounds and morin.

[00107] Figure 1 1A-D depicts the effect on tau aggregation of synthesized bi-aromatic compounds and nicotinic acid and the modulation, or lack thereof, of tau aggregation, seen as an increased initial rate of tau aggregation but reduced equilibrium or plateau level of aggregation, by compounds of the current invention, as shown in Example 8, as measured by ThS fluorescence.

[00108] Figure 12A-B depicts inhibition of a-synuclein aggregation by compounds of the present invention as shown in Example 8, as measured by ThT
fluorescence.

[00109] Figure 13A-B depicts the mean (- SE) change in primary efficacy variables from baseline in a human clinical trial of L-Trp in people with AD;
*
p<0.001, f p<0.01, from Example 16.

[00110] Figure 14 depicts "typical" binding of L-Trp to HHQK region of A,6 wherein it is shown binding to His13 and Lys 16 of PDB structure 1AML as discussed in Example 17.

[00111] Figure 15 depicts alternative binding of L-Trp to HHQK region of A(3 wherein it is shown occurring to His14 and Lys 16 of PDB structure 1BA4 as discussed in Example 17.

RECTIFIED SHEET (RULE 91) [00252] Figure 16 depicts the interaction of Oc with KREH receptor of B7-1 as discussed in Example 18.

[00253] Figure 17 depicts the interaction of Oc with RDHH receptor of ICAM-I as discussed in Example 18.

[00254] Figure 18 depicts the interaction of Oc with HKEK receptor of IL-1R1 as discussed in Example 18.

DETAILED DESCRIPTION

[00255] The compounds of the present invention can be administered to anyone requiring treatment of a protein folding disease or systemic amyloidoses. For example, the compounds are useful for treating Alzheimer's disease, for helping prevent or delay the onset of Alzheimer's disease, for treating patients with MCI (mild cognitive impairment) and preventing or delaying the onset of Alzheimer's disease in those who would progress from MCI to AD, for treating Down's syndrome, for treating humans who have Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-Type, for treating cerebral amyloid angiopathy and preventing its potential consequences, i.e. single and recurrent lobal hemorrhages, for treating other degenerative dementias, including dementias of mixed vascular and degenerative origin, dementia associated with Parkinson's disease, dementia associated with progressive supranuclear palsy, dementia associated with cortical basal degeneration, dementia associated with tauopathies, and diffuse Lewy body type Alzheimer's disease. Preferably, the compounds and compositions of the invention are particularly useful for treating or preventing Alzheimer's disease.

[00256] Di- and polyanionic sulfate and sulfonate compounds have been shown to inhibit in vitro aggregation of amyloidogenic proteins, including the Alzheimer peptide, A(3 (Kisilevsky et al., Nat. Med., 1:143-8, 1995). It is thought that these anionic compounds in vivo would inhibit A(3 deposition by disrupting A(3-glycosaminoglycan.

[00257] It is believed that the compounds and methods of the present invention will result in a therapeutic outcome by binding the His13-His14-G1nj5-Lys16 region of A[3 via cation-7E interactions, rather than cationic-anionic interactions.
Without being bound by theory, it is believed that the compounds of the present invention containing two aromatic groups would form cation-7c interactions at two of the three cationic residues in the His13-His14-Glnjs-Lys16 region and thereby interfere with A(3 aggregation (See, The HHQK Domain of (3-Amyloid Provides a Structural Basis for the Immunopathology of Alzeheimer's Disease, The Journal of Biological Chemistry, Vol. 274, No. 45, pp 29719-29726, 1988).

[00258] The compounds of certain embodiments of the invention are non-peptidic, small organic molecules. Because of this, they are expected to overcome deficiencies of peptidic compounds such as poor pharmacokinetics, e.g., degradation by proteases.

[00259] When treating or preventing these diseases, the compounds of the invention can either be used individually or in combination. For example, aministration may be orally, topically, by suppository, inhalation, subcutaneously, intravenously, bucally, sublingually, or parenterally.

[00260] Various oral dosage forms can be used, including such solid forms as tablets, gelcaps, capsules, caplets, granules, lozenges and bulk powders and liquid forms such as, e.g., emulsions, solution and suspensions. The compounds of the present invention can be administered alone or can be combined with various pharmaceutically acceptable carriers and excipients known to those skilled in the art, including but not limited to diluents, suspending agents, solubilizers, binders, disintegrants, preservatives, coloring agents, lubricants and the like.

[00261] When the compounds of the present invention are incorporated into oral tablets, such tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, multiply compressed or multiply layered. Liquid oral dosage forms include aqueous and nonaqueous solutions, emulsions, suspensions, and solutions and/or suspensions reconstituted from non-effervescent granules, containing suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, coloring agents, and flavoring agents. When the compounds of the present invention are to be injected parenterally, they may be, e.g., in the form of an isotonic sterile solution. Alternatively, when the compounds of the present invention are to be inhaled, they may be formulated into a dry aerosol or may be formulated into an aqueous or partially aqueous solution.

[00262] In addition, when the compounds of the present invention are incorporated into oral dosage forms, it is contemplated that such dosage forms may provide an immediate release of the compound in the gastrointestinal tract, or alternatively may provide a controlled and/or sustained release through the gastrointestinal tract. A wide variety of controlled and/or sustained release formulations are well known to those skilled in the art, and are contemplated for use in connection with the formulations of the present invention. The controlled and/or sustained release may be provided by, e.g., a coating on the oral dosage form or by in-corporating the compound(s) of the invention into a controlled and/or sustained release matrix.

[00263] Specific examples of pharmaceutically acceptable carriers and excipients that may be used to formulate oral dosage forms, are described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986). Techniques and compositions for making solid oral dosage forms are described in Pharmaceutical Dosage Forms: Tablets (Lieberman, Lachman and Schwartz, editors) 2nd edition, published by Marcel Dekker, Inc. Techniques and compositions for making tablets (compressed and molded), capsules (hard and soft gelatin) and pills are also described in Remington's Pharmaceutical Sciences (Arthur Osol, editor), 1553B 1593 (1980). Techniques and composition for making liquid oral dosage forms are described in Pharmaceutical Dosage Forms: Disperse Systems, (Lieberman, Rieger and Banker, editors) published by Marcel Dekker, Inc.

[00264] When the compounds of the present invention are incorporated for parenteral administration by injection (e.g., continuous infusion or bolus injection), the formulation for parenteral administration may be in the form of suspensions, solutions, emulsions in oily or aqueous vehicles, and such formulations may further comprise pharmaceutically necessary additives such as, e.g., stabilizing agents, sus-pending agents, dispersing agents, and the like. The compounds of the invention may also be in the form of a powder for reconstitution as an injectable formulation.
[00265] The compounds and compositions of the invention can be enclosed in multiple or single dose containers. The enclosed compounds and compositions can be provided in kits, for example, including component parts that can be assembled for use. The kit can also optionally include instructions for use in any medium.
For example, the instructions can be in paper or electronic form. For cxample, a compound of the present invention in lyophilized form and a suitable diluent may be provided as separated components for combination prior to use. A kit may include a compound of the present invention and a second therapeutic agent for co-administration. The compound of the present invention and second therapeutic agent may be provided as separate component parts. A kit may include a plurality of containers, each container holding one or more unit dose of the compound of the invention. The containers are preferably adapted for the desired mode of administration, including, but not limited to tablets, gel capsules, sustained-release capsules, and the like for oral administration; depot products, pre-filled syringes, ampules, vials, and the like for parenternal administration; and patches, medipads, creams, and the like for topical administration.

[00266] The concentration of active compound in the drug composition will depend on absorption, inactivation, and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.

[00267] The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

[00268] The compounds of the invention can be used in combination, with each other or with other therapeutic agents or approaches used to treat or prevent the protein folding conditions described abovc. Such agents include, for example, cholinesterase inhibitors (such as, e.g., acetylcholinesterase inhibitors and butyrylcholinesterase inhibitors); gamma-secretase inhibitors; beta-secretase inhibitors; anti-inflammatory agents; anti-oxidants; immunological approaches;
NMDA antagonists; cholesterol lowering agents (such as, e.g., statins); and direct or indirect neurotropic agents.

[00269] Acetylcholinesterase inhibitors include compounds such as, e.g., tacrine (tetrahydroaminoacridine, marketed as Cognex ), donepezil hydrochloride, (marketed as Aricept ), rivastigmine (marketed as Exelon(&) and galantamine (Reminyl ).

[002701 Anti-oxidants include compounds such as, e.g., tocopherol, ascorbic acid, beta carotene, lipoic acid, selenium, glutathione, cysteine, coenzyme Q, vitamin E and ginkolides.

[00271] NMDA (N-methyl-D-aspartate) antagonists include, for example, memantine (Namenda ).

[00272] Immunological approaches include, for example, immunization with beta-amyloid peptides (or fragments thereof) or administration of anti-beta-amyloid antibodies.

[00273] Direct or indirect neurotropics agents include, for example, Cerebrolysin and AIT-082 (Emilieu, 2000, Arch. Neurol. 57:454).

[00274] Anti-inflammatory agents include, for example, Cox-II inhibitors such as, e.g., rofecoxib, celecoxib, DUP-697, flosulide, meloxicam, 6-MNA, L-745337, nabumetone, nimesulide, NS-398, SC-5766, T-614, L-768277, GR-253035, JTE-522, RS-57067-000, SC-58125, SC-078, PD-138387, NS-398, flosulide, D-1367, SC-5766, PD-164387, etoricoxib, valdecoxib, parecoxib and pharmaceutically acceptable salts thereof. Other anti-inflammatory agents include, for example, aspirin, ibuprofen, diclofenac, naproxen, benoxaprofen, flurbiprofen, fenoprofen, flubufen, ketoprofen, indoprofen, piroprofen, carprofen, oxaprozin, pramoprofen, muroprofen, trioxaprofen, suprofen, aminoprofen, tiaprofenic acid, fluprofen, bucloxic acid, indomethacin, sulindac, tolmetin, zomepirac, tiopinac, zidometacin, acemetacin, fentiazac, clidanac, oxpinac, mefenamic acid, meclofenamic acid, flufenamic acid, niflumic acid tolfenamic acid, diflurisal, flufenisal, piroxicam, sudoxicam, isoxicam and pharmaceutically acceptable salts thereof.

[00275] Statins include, for example, atorvastatin, simvastatin, pravastatin, cerivastatin, mevastatin, velostatin, fluvastatin, lovastatin, dalvastatin, rosuvastatin, fluindostatin, dalvastain and pharmaceutically acceptable salts thereof.

[00276] Other cholesterol reducing compounds include bile sequestration compounds (e.g., colestipol and cholestyramine); fibrin (e.g., gemfibrozil, fenofibrate, psyllium, wheat bran, oat bran, rice bran, corn bran, konjak flour, Jerusalem artichoke flour, fruit fiber and any other functional food products) and other agents such as, e.g., nicotinic acid (niacin).

[002771 In addition, the compounds of the invention can also be used with inhibitors of P-glycoprotein (P-gp). The use of P-gp inhibitors is known to those skilled in the art. See for example, Cancer Research, 53, 4595-4602 (1993), Clin.
Cancer Res., 2, 7-12 (1996), Cancer Research, 56, 4171-4179 (1996), International Publications W099/64001 and WO01/10387. P-gp inhibitors are useful by inhibiting P-gp from decreasing brain blood levels of the compounds of the invention.
Suitable P-gp inhibitors include cyclosporin A, verapamil, tamoxifen, quinidine, Vitamin E-TGPS, ritonavir, megestrol acetate, progesterone, rapamycin, 10,11-methanodibenzosuberane, phenothiazines, acridine derivatives such as, e.g., GF120918, FK506, VX-710, LY335979, PSC-833, GF-102,918 and other steroids.

[00278] All of the additional agents disclosed above may be administered at the same or different time and/or route of administration than the compounds of the present invention.

[00279] The following examples illustrate various aspects of the present invention, and are not to be construed to limit the claims in any manner whatsoever.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[00280] The following examples refer to compounds listed in Table 1 below:
Table 1 R~~ I ~ / I R R I~ I CHz I \ I R' M H\ H H

R R' n R R' Oa H H 1a 1 H H
Ob OCH3 5-OCH3 ic 1 OCH3 OCH3 Oc Br 5-CO2H le I H COZH
Od H 5-CO2H 1f 1 CO2H COZH
Oe OCH3 5-CO2H 1g I OH OH
Of F 5-CO2H 2a 2 H H
Og CI 5-CO2H 2b 2 Br CO2H
Oh CH3 5-CO2H 2c 2 CO2H CO2H
0i OCF3 5-CO2H 3 3 H H
Oj OH 5-CO2H 4 4 H H
Ok H 6-CO2H 5 5 H H

Om OH 5-OH 7 p-xylyl H H
R N
HO
H
H OH
R
R R' N
1i H
1b H H
Id OCH3 OCH3 HO
I / ~ H
N
HO I~ I I / I OH H I ~ I

/ N N OH
H H
1h ij Synthesis of Directly Linked Bis-Indoles [00281] Directly linked bis-indoles were synthesized according to the procedure set forth in Scheme 1 below:

O OH
R I\ R I/\ I I\ R
/ H O EtOH, 45 -~ 25 C H O

8 Piperidine (cat.) 9a,c-I
+ 8-24 h rs.
~ BH3-THF THF
R'- I I 12-20 hrs.
~ N
H
R
I /, I I I ~ R, N N
H
R R' Oa H H
NaOMe/MeOH Ob OCH3 5-OCH3 CuI, DMF 10c Br 5-Br Reflux, 18 h 10d H 5-Br \ -- ; , 10e Br 5-OCH3 10f F 5-Br 10g Cl 5-Br 10h Br 5-CH3 10i OCF3 5-Br 10j Br 5-OH
10k H 6-Br 101 H 7-Br Scheme 1, Synthesis of 3,3'-bis-indolyl compounds (i) Coupling of indole and isatin (9a, c-I) [00282] Following the procedure of Bergman (J. Acta. Chem. Scand., 1971, 4:
1277-80) a solution of isatin (5-20 mmol) and indole (1 eq.), either or both substituted at the 5, 6 or 7 position as appropriate (Scheme 1), and piperidine (0.1 eq.) were stirred in ethanol at 45 C for one hour and then at room temperature. When TLC
indicated the reaction was complete (8-24 hrs.), the reaction mixture was filtered in cases where a small amount of solid was present, and the product purified by recrystallization from EtOHIH20 (9a,d,f,j), THF/EtOH/H2O (9g,h), or EtOAc/hexanes (9i). For 9c and 9e, purification was achieved by removing the solvent from the reaction mixture, resuspending the yellow solid in EtOH (50 mL), sonicating for 2 hrs. to dissolve impurities, collecting the solid by vacuum filtration, and rinsing with EtOH.

[002831 All NMR chemical shift data presented herein is in ppm (parts per million) and J coupling contants are presented herein in Hertz. Due to symmetry, some 13C carbon peaks correspond to 2 carbons, where indicated by (2c).

3-Hydroxy-3-(indol-3 yl)-indolin-2-one (9a):

[00284] Light beige crystals (7.47g, 94%); mp 120 C (dec.), lit. 123 C (dec.) (Berens U et al. 1996. Tetrahedron Asymmetry 7:285-92); 'H NMR: 6.33 (s, IH), 6.88 (m, 2H), 6.95 (t, IH, J=7.5), 7.02 (t, 1H, J=7.5), 7.06 (d, IH, J=2.5), 7.23 (m, 2H), 7.32 (d, 1H, J=8.3), 7.35 (d, 1H, J=8.2), 10.31 (s, IH), 10.96 (s, 1H);13C NMR:
74.87, 109.56, 111.44, 115.41, 118.41, 120.29, 121.00, 121.63, 123.47, 124.72, 124.89, 128.99, 133.42, 136.77, 141.64, 178.41; El (electron impact) m/z (%):
264.1 (25), 247.4 (47), 117.2 (37), 28.2 (100); HRMS (High Resolution Mass Spectroscopy): C16H12N202 requires 264.0899, found 264.0897.

5-bromo-3-(5-bromo-indol-3-yl)-3-hydroxy-indolin-2-one (9c):

[00285] Light yellow powder (5.40 g, 71%); mp 160 C (dec.).1H NMR: 6.57 (s, IH), 6.88 (d, IH, J=8.2), 7.01 (d, 1 H, J=2.5), 7.18 (dd, IH, J=8.5, J=1.9), 7.3 5(m, 2H), 7.47 (dd, 1H, J=8.4, J=2.1), 7.75 (d, IH, J=1.9), 10.49 (s, 1H), 11.24 (s, 1H);13C
NMR: 74.66, 111.27, 111.78, 113.39, 113.63, 114.48, 122.97, 123.72, 125.12, 126.74, 127.39, 131.91, 135.24, 135.55, 140.92, 177.64.

3-(5-bromo-indol-3 yl)-3-hydoxy-indolin-2-one (9d):

[00286] Light orange crystals (5.55 g, 81%); mp 118 C (dec.); 'H NMR: 6.41 (s, 1H), 6.90 (d, 1H, J=7.5), 6.98 (d, 1H, J=2.5), 7.00 (t, 1H, J=7.6), 7.16 (dd, 1H, J=8.6, J=2.1), 7.27 (m, 2H), 7.32 (d, 1H, J=8.6), 7.71 (d, 1H, J=1.8), 10.33 (s, 1H), 11.18 (s, 1H); 13C NMR: 74.61, 109.64, 111.13, 113.49, 115.22, 121.76, 123.11, 123.56, 124.74, 125.04, 126.90, 129.20, 132.84, 135.50, 141.61, 178.16.

5-bromo-3-hydroxy-3-(5-methoxy-indol-3 yl)-indolin-2-one (9e):

[00287] Beige powder (0.773 g, 45%); mp 214 C (dec.);1H NMR: 3.64 (s, 3H), 6.48 (s, 1H), 6.71 (dd, 1H, J=8.8, J=2.5), 6.85 (d, IH, J=2.4), 6.88 (d, 1H, J=8.3), 7.03 (d, 1 H, J=2.6), 7.24 (d, IH, J=8.8), 7.34 (d, 1H, J=2.0), 7.44 (dd, 1 H, J=8.2, J=2.1), 10.47 (s, IH), 10.88 (d, 11-I, J=1.7); 13C NMR: 55.15, 74.91, 102.38, 110.93, 111.63, 112.13, 113.27, 114.20, 124.24, 125.10, 127.37, 131.67, 131.96, 135.71, 140.93, 152.81, 177.82.

3-(5-bromo-indol-3 yl)-5 fluoro-3-hydoxy-indolin-2-one (9f):

[00288] Beige powder (5.37 g, 74%); mp 211 C (dec.); 'H NMR: 6.55 (s, 1H), 6.90 (dd, 1H, J=8.3, J=4.2), 7.01 (d, 1H, J=2.4), 7.11 (m, 2H), 7.17 (dd, 1H, J=8.6, J=2.0), 7.32 (d, 1H, J=8.6), 7.75 (d, 1H, J=1.8), 10.36 (s, 1H), 11.23 (s, 1H);13C

NMR: 74.91, 110.52, 110.58, 111.23, 112.23, 112.43, 113.57, 114.61, 115.38, 113.57, 123.10, 123.68, 125.15, 126.81, 134.50, 134.55, 135.54, 137.76, 157.09, 158.98, 178.10.

3-(5-bromo-indol-3 yl)-5-chloro-3-hydoxy-indolin-2-one (9g):

[00289] Light orange solid (5.59 g, 74%); mp 215 C (dec.);'H NMR: 6.58 (s, IH), 6.92 (d, 1H, J=8.3), 7.01 (d, IH, J=2.5), 7.18 (dd, IH, J=8.6, J=1.9), 7.26 (d, IH, J=2.1), 7.3 4(m, 2H), 7.75 (d, IH, J=1.8), 10.49 (s, 1 H), 11.24 (d, 1 H, J=1.4); 13C
NMR: 74.64, 111.178, 111.20, 113.55, 114.39, 122.94, 123.65, 124.61, 125.07, 125.68, 126.69, 129.00, 134.77, 135.48, 140.43, 177.71.
5-bromo-3-hydroxy-3-(5-methyl-indol-3-yl)-indolin-2-one (9h):

[00290] Yellow solid (2.13 g, 86%); mp 205 (dec.);'H NMR: 2.29 (s, 3H), 6.46 (s, 1H), 6.88 (m, 2H), 7.00 (d, 1H, J=2.4), 7.23 (m, 2H), 7.32 (d, 1H, J=1.9), 7.43 (dd, 1H, J=8.3, J=2.1), 10.47 (s, 1H), 10.89 (d, 1H, J=1.6); 13C NMR: 21.31, 74.86, 111.20, 111.59, 113.17, 114.07, 119.71, 122.69, 123.46, 124.91, 126.77, 127.21, 131.55, 135.09, 135.81, 140.81, 177.83.

3-(5-bromo-indol-3 yl)-3-hydoxy-5-(trifluoromethoxy)-indolin-2-one (9i):

[00291] Light beige powder (0.802 g, 41%); mp 201 C (dec.); 'H NMR: 6.62 (s, 1 H), 6.97 (s, 1 H), 6.99 (d, 1 H, J=7.4), 7.18 (dd, 1 H, J=8.6, J=1.9), 7.24 (s, 1 H), 7.32 (m, 2H), 7.73 (d, 1H, J=1.6), 10.53 (s, IH), 11.25 (s, 1H);13C NMR:
74.75, 110.71, 111.31, 113.65, 114.36, 118.27, 122.53, 123.09, 123.78, 125.16, 126.78, 134.40, 135.54, 140.80, 143.23, 178.06.

3-(5-Hydroxy-indol-3-yl)-5-bromo-3-hydroxy-indoline-2-one (9j):

[00292] Light beige solid (1.85 g, 57%); mp 215 C (dec.); 'H NMR S: 6.41 (s, 1 H), 6.57 (dd, 1 H, J=8.7, J=2.3), 6.75 (d, 1 H, J=2.2), 6.86 (d, 1 H, J=8.2), 6.98 (d, 1 H, J=2.6), 7.13 (d, 1H, J=8.6), 7.30 (d, 1H, J=2.0), 7.42 (dd, IH, J=8.2, J=2.1), 8.57 (s, 1H), 10.45 (s, 1H), 10.71 (d, 1H, J=2.0);13C NMR 5: 74.89, 104.23, 111.55, 111.66, 111.74, 113.20, 113.66, 123.79, 125.38, 127.16, 131.27, 131.54, 135.90, 140.85, 150.15, 177.90.

3-(6-Bromo-indol-3-yl)-3-hydoxy-indolin-2-one (9k):

[00293] Light beige crystals (1.46 g, 84%); mp 182 C (dec.); 'H NMR S: 6.39 (s, 1H), 6.89 (d, 1H, J=7.4), 6.97 (dt, 1H, J=7.5, J=0.9), 7.02 (d, 1H, J=2.5), 7.04 (dd, 1H, J=8.5, J=1.8), 7.25 (m, 2H), 7.41 (d, 1H, J=8.5), 7.53 (d, IH, J=1.8), 10.32 (s, 1H), 11.10 (d, 1H, J=1.6); 13C NMR 8: 74.62, 109.63, 113.85, 113.99, 115.76, 121.33, 121.69, 122.33, 124.05, 124.48, 124.68, 129.11, 133.00, 137.66, 141.58, 178.15.
3-(7-Bromo-indol-3-yl)-3-hydoxy-indolin-2-one (91):

Yellow solid (1.41 g, 88%); mp 190 C (dec.); 'H NMR S: 6.44 (s, 1H), 6.85 (t, IH, J=7.8), 6.91 (d, 1H, J=7.7), 6.97 (t, 1H, J=7.5), 7.07 (d, 1H, J=2.6), 7.26 (m, 3H), 7.39 (d, 1H, J=8.0), 10.36 (s, 1H), 11.21 (d, 1H, J=1.8); 13C NMR S: 74.68, 104.06, 109.68, 116.93, 119.93, 119.97, 121.74, 123.66, 124.58, 124.71, 126.60, 129.20, 132.93, 134.96, 141.63, 178.07.

(ii) Reduction to bis-indolyl species (0a, lOc-1) [00294] To a solution of 9 (4-16 mmol) in dry THF at 0 C was added BH3=TIlF
(1.0 M, 2.5 eq.) dropwise over 10 min., causing the solution to turn from light yellow to orange. The solution was stirred at room temperature overnight, then quenched by the dropwise addition of MeOH (30mL) over 10 min., causing the solution to change from orange back to yellow. The trimethyl borate formed in this step and the solvent were removed under vacuum. The yellow solid was washed with MeOH (30 mL), which was removed under vacuum along with any remaining trimethyl borate. The solid was recrystallized and/or purified by flash column chromatography, as indicated.
3,3'-bis-indolyl (0a):

[00295] Recrystallized from THF/CH2C12. Yellow or green crystals (3.61g, 57%); mp 285-287 C (lit. 285-287 C [120]); 'H NMR: 7.05 (t, 2H, J=7.1), 7.14 (t, 2H, J=7.4), 7.43 (d, 2H, J=8.0), 7.62 (d, 2H, J=2.3), 7.77 (d, 2H, J=8.0), 11.13 (s, 2H);
13C NMR: 110.62, 112.44, 119.67, 120.46, 122.06, 122.72, 126.97, 137.28; El m/z (%): 232 (100); HRMS: C16Hj2N2 requires 232.1000, found 232.1005.
5-bromo-3-(5-bromo-indol-3-yl)-indole (lOc):

[00296] Purification by flash chromatography in 2:1 hexanes:EtOAc and subsequent recrystallization of impure fractions from MeOH/1-120. White or yellow crystals (2.25 g, 58%); mp 201-203 C; 'H NMR: 7.27 (dd, 2H, J=8.6, J=1.8), 7.45 (d, 2H, J=8.5), 7.77 (d, 2H, J=2.2), 7.88 (s, 2H), 11.45 (s, 2H); 13C NMR: 108.64, 111.62, 113.62, 121.41, 123.79, 123.88, 127.73, 135.04; El m/z (%): 392 (89), 390 (100), 388 (31), 310 (23), 195 (35); HRMS: C16HIpBrzN2 requires 387.9211, found 387.9222.

5-bromo-3-(indol-3 yl)-indole (10d):

[00297] Recrystallization from EtOH, followed by flash chromatography of the filtrate (after reducing the volume) in 3:1 hexanes:EtOAc. Yellow solid (1.78g, 48%); mp 206 C (dec.);'H NMR: 7.07 (t, IH, J=7.1), 7.15 (t, IH, J=7.5), 7.26 (dd, 1 H, J=8.6, J=1.8), 7.42 (d, 1 H, J=8.6), 7.45 (d, 1 H, J=8.1), 7.66 (d, 1 H, J=2.3), 7.70 (d, IH, J=2.3), 7.74 (d, IH, J=7.9), 7.87 (s, 1H), 11.18 (s, IH), 11.38 (s, IH);13C
NMR: 108.80, 109.49, 111.43, 111.59, 113.54, 118.87, 119.31, 121.23, 121.56, 122.16, 123.51, 123.65, 125.91, 127.82, 135.00, 136.34.

5-bromo-3-(5-methoxy-indol-3-yl)-indole (l0e):

[00298] Recrystallization from EtOH/THP, followed by flash chromatography of the filtrate (after reducing the volume) in 2:1 hexanes:EtOAc. Beige solid (0.773 g, 45%); mp 214 C (dec.); 'H NMR: 3.77 (s, 3H), 6.80 (dd, IH, J=8.7, J=2.3), 7.16 (d, 1H, J=2.2), 7.25 (dd, 1H, J=8.6, J=1.8), 7.34 (d, IH, J=8.7), 7.42 (d, 1H, J=8.6), 7.60 (d, 1H, J=2.4), 7.69 (d, 1H, J=2.3), 7.85 (d, 1H, J=1.6), 11.03 (s, 1H), 11.35 (s, 1H);
13C NMR: 55.29, 101.08, 108.59, 109.59, 111.35, 111.41, 112.24, 113.53, 121.59, 122.90, 123.40, 123.60, 126.18, 127.83, 131.49, 135.00, 153.42.

5-bromo-3-(5 fluoro-indol-3 yl)-indole (10f):

[00299] Purification by flash chromatography in 3:1 hexanes:EtOAc. Grey powder (0.940 g, 48%); mp 154-156 C;'H NMR: 7.00 (dt, 1H, J=9.1 (t), J=2.5), 7.26 (dd, 1H, J=8.6, J=1.9), 7.44 (m, 3H), 7.74 (d, 1H, J 2.4), 7.77 (d, 1H, J=2.4), 7.87 (d, 1H, J=1.8), 11.30 (s, IH), 11.41 (s, 1H) ;13C NMR: 103.79, 103.98, 109.07, 109.11, 109.26, 109.47, 111.47, 112.43, 112.51, 113.51, 121.40, 123.54, 123.67, 124.21, 125.89, 125.97, 127.60, 132.96, 134.95, 156.13, 157.97.

5-bromo-3-(5-chloro-indol-3-yl)-indole (10g):

[003001 Purification by flash chromatography in 3:1 hexanes:EtOAc. Light green solid (1.35 g, 65%); mp 198-200 C;'H NMR: 7.15 (dd, 1H, J=8.6, J=2.0), 7.26 (dd, iH, J=8.6, J=1.9), 7.43 (d, 1H, J=8.6), 7.47 (d, IH, J=8.6), 7.73 (d, IH, J=2.0), 7.76 (d, 1H, J=2.4), 7.77 (d, 1H, J=2.4), 7.87 (d, 1H, J=1.8), 11.41 (s, IH), 11.43 (s, 1H); 13C NMR: 108.61, 108.67, 111.53, 113.08, 113.55, 118.34, 121.20, 121.35, 123.59, 123.72, 123.78, 123.98, 126.92, 127.63, 134.75, 134.97.
5-bromo-3-(5-methyl-indol-3 yl)-indole (10h):

[00301] Purification by flash chromatography in 3:1 hexanes:EtOAc. Yellow powder (0.769 g, 47%); mp 214-216 C;'H NMR: 2.41 (s, 3H), 6.97 (d, 1H, J=8.3), 7.25 (d, 1H, J=8.3), 7.34 (d, IH, J=8.1), 7.42 (d, 1H, J=8.5), 7.54 (s, IH), 7.61 (s, IH), 7.70 (s, 1H), 7.87(s, 1H), 11.04 (s, 1H), 11.36 (s, 1H);'3C NMR: 21.34, 108.30, 109.65, 111.29, 111.43, 113.53, 118.93, 121.59, 122.24, 122.88, 123.47, 123.63, 126.18, 127.34, 127.88, 134.73, 135.03.

5-bromo-3-(5-(trffluoromethoxy)-indol-3 y1)-indole (10i):

[00302] Purification by flash chromatography in 2:1 hexanes:EtOAc. Yellow powder (0.450 g, 63%); mp 125-127 C;'H NMR: 7.12 (dd, 1H, J=8.8, J=1.0), 7.26 (dd, 1H, J=8.6, J=1.9), 7.43 (d, 1H, J=8.6), 7.53 (d, IH, J=8.7), 7.62 (s, 1H), 7.72 (d, 1H, J=2.3), 7.81 (d, 1H, J=2.3), 7.84 (d, 1H, J=1.7), 11.42 (s, 1H), 11.49 (s, 1H);13C
NMR: 108.61, 109.49, 111.61, 112.71, 113.69, 114.96, 117.47, 119.49, 121.44, 121.52, 123.54, 123.83, 123.84, 124.71, 125.94, 127.70, 134.85, 135.07, 141.87, 141.88.

3-(5-Bromo-indol-3 yl)-indol-5-ol (lOj):

[003031 Purification by flash chromatography in 1:1 hexanes:THF. Yellow solid (0.560 g, 33%); mp 183 C (dec.); 'H NMR S: 6.67 (dd, IH, J=8.6, J=2.2), 7.02 (d, 1 H, J=2.1), 7.23 (m, 2H), 7.41 (d, i H, J=8.6), 7.51 (d, 1 H, J=2.4), 7.55 (d, 1 H, J=2.3), 7.81 (d, 1H, J=0.9), 8.63 (s, IH), 10.86 (s, 1H), 11.32 (s, 1H); 13C
NMR S:
103.14, 107.86, 109.85, 111.24, 111.50, 111.83, 113.45, 121.55, 122.63, 123.05, 123.51, 126.64, 127.83, 130.81, 134.89, 150.66.

6-Bromo-3-(indol-3-yl)-indole (10k):

[00304] Hot filtration and recrystallization from EtOH. Yellow crystals (0.578 g, 47%); mp 238 C (dec.); 'H NMR S: 7.06 (t, 1H, J=7.3), 7.15 (t, 1H, J=7.4), 7.18 (dd, IH, J=8.5, J=1.8), 7.45 (dd, 1H, J=8.1, J=0.7), 7.62 (m, 1H), 7.64 (d, 1H, J=2.0), 7.67 (d, 1H, J=1.7), 7.72 (d, IH, J=8.5), 7.76 (d, 1H, J=7.9), 11.19 (s, 1H), 11.29 (s, 1H); 13C NMR 5: 109.43, 110.51, 112.01, 114.33, 114.47, 119.32, 119.87, 121.69, 121.77, 122.04, 122.50, 123.23, 125.50, 126.33, 136.79, 137.64.
7-Bromo-3-(indol-3-yl)-indole (101):

[00305] Suspended solid in MeOH (20 mL) and sonicated for 1 hr., filtered and rinsed with MeOH. Yellow solid (0.831 g, 67%); mp 240 C (dec.); 'H NMR S: 7.02 (t, 1 H, J=7.7), 7.0 8(t, 1 H, J=7.1), 7.15 (t, 1 H, J=7.1), 7.3 7(d, 1 H, J=7.4), 7.45 (d, 1 H, J=8.1), 7.65 (m, 2H), 7.73 (d, 1H, J=7.9), 7.78 (d, 1H, J=7.9), 11.21 (s, 1H), 11.38 (s, 1H); 13C NMR 8: 104.35, 180.90, 111.18, 111.58, 118.93, 119.15, 119.32, 120.18, 121.26, 122.29, 122.96, 123.80, 125.93, 127.84, 134.58, 136.33.

(iii) Conversion of 5-bromo-3-(5-bromo-indol-3-yl)-indole (10c) to 5-methoxy-3-(5-methoxy-indol-3-y!)-indole (0b) [003061 As depicted in Scheme 1(iii), to a solution of tOc (0.780 g, 2.0 mmol) in DMF (8 mL) was added NaOMe (25 wt.% in MeOH, 9.0 mL, 20 eq.) and CuI (1.53 g, 4 eq.). After heating the suspension at reflux for 18 hrs., NH3 (aq., 15 mL) was added and the aqueous layer extracted three times with EtOAc (10 mL). The organic layer was dried with MgSO4, concentrated to -1 mL and purified by flash column chromatography using 2:1 hexanes:EtOAc as the solvent system, giving Ob as a grey powder (0.108 g, 19%).

5-methoxy-3-(5-methoxy-indol-3-yl)-indole (0b) [00307] Grey powder (0.108 g, 19%); mp 193-196 C; 'H NMR: 3.32 (s, 6H), 6.78 (dd, 2H, J=8.7, J=2.2), 7.17 (d, 2H, J=2.0), 7.33 (d, 2H, J=8.7), 7.55 (d, 2H, J=2.2), 10.95 (s, 2H); 13C NMR: 101.12, 109.53, 111.30, 112.10, 122.44, 126.29, 131.44, 153.25; El m/z (%): 292 (100), 262 (5.5); HRMS: C18H16N202 requires 292.1212, found 292.1223.

(iv) Carboxylation of bromo-bis-indolyl species (2b, 2c, Oc-1) 1) KH, THF, 0 C

2) t-BuLi, -78 C

10c-t 10 Oc-I
3) COZ(s), -78 C

Scheme 2, Carboxylation of 3,3'-bis-indolyl compounds [00308] As depicted in Scheme 2, the bromo-indole species (0.8 - 3.0 mmol) was dissolved in dry THF (10 mL) and added dropwise to a suspension of KH (2.2 eq., 35 wt.% in oil) in THF (20 mL) at 0 C. The addition usually caused the reaction mixture to turn dark blue, regardless of initial colour. After 20 min., the reaction was cooled to -78 C and t-BuLi (3 eq., 1.7M in pentane) was added dropwise, causing the reaction to turn a butterscotch colour. After a further 20 min. of stirring, a large excess of dry ice was added. The reaction was quenched after a final 20 min.
period of stirring by adding MeOH (5 mL), followed by water (10 mL) and HCI (IN) until a pH of 2 was reached. The aqueous layer was extracted with EtOAc (2 x 20 mL) and the organic layer dried with Na2SO4. Products were purified by flash column chromatography using, as eluents, hexanes/EtOAc or hexanes/THF, both containing 2-5% AcOH. Other than Oe, products were isolated as 1:1 adducts with AcOH.
3-(5-bromo-indol-3 yl)-indole-5-carboxylic acid (0c) = AcOH

[00309] Purification by flash chromatography using 2:1 hexanes:EtOAc, 2%
AcOH. Beige solid (0.138 g, 39%); mp 225 C (dec.); 1 H NMR: 1.91 (s, 3H), 7.26 (dd, 1 H, J=8.6, J=1.7), 7.45 (d, 1H, J=8.6), 7.50 (d, 1 H, J=8.5), 7.69 (d, 1 H, J=2.3), 7.77 (m, 214), 7.85 (s, IH), 8.38 (s, 1H), 11.44 (s, 1H), 11.56 (s, 1H), 12.19 (bs, 2H);
13C NMR: 20.95, 108.64, 110.08, 111.29, 111.57, 113.62, 121.36, 121.44, 121.86, 122.50, 123.74, 123.77, 123.83, 125.58, 127.76, 134.99, 138.71, 168.31, 171.87; El m/z (%): 355 (0.9), 312 (100), 310 (73); HRMS: C17H11BrNZOZ requires 355.0004, found 355.0010.

3-(indol-3 yl)-indole-5-carboxylic acid (Od) = AcOH

[00310] Purification by flash chromatography using 2:1 hexanes:THF, 5%
AcOH. Yellow powder (0.510 g, 60%); mp 226 C (dec.); 1H NMR: 1.91 (s, 3H), 7.07 (t, 1 H, J=7.1), 7.16 (t, 1 H, J=7.1), 7.47 (d, 1 H, J=8.1), 7.50 (d, 1 H, J=8.5), 7.61 (d, IH, J=2.4), 7.71 (d, 1H, J=2.3), 7.74 (d, 1H, J=7.9), 7.77 (dd, 1H, J=8.5, J=1.5), 8.41 (s, 1H), 11.20 (s, 1H), 11.52 (s, 11-1), 12.24 (bs, 2H); 13C NMR: 20.98, 108.86, 110.97, 111.20, 111.57, 118.86, 119.29, 121.24, 121.33, 122.11, 122.13, 122.41, 123.32, 125.68, 125.96, 136.31, 138.70, 168.39, 171.91; El m/z (%): 276.0 (100) ;
HRMS:
C17H12N202 requires 276.0899, found 276.0902.
3-(5-methoxy-indol-3-yl)-indole-5-carboxylic acid (0e) [00311] Purification by flash chromatography using 1.6:1 hexanes:THF, 5%
AcOH. Beige powder (0.128 g, 36%); mp 170 C (dec.);1H NMR: 3.76 (s, 3H), 6.81 (dd, 1H, J=8.7, J=2.4), 7.18 (d, 1H, J=2.3), 7.36 (d, 1H, J=8.7), 7.50 (d, 1H, J=8.5), 7.56 (d, IH, J=2.4), 7.71 (d, 1H, J=2.2), 7.77 (dd, 1H, J=8.5, J=1.5), 11.06 (s, 1H), 11.49 (s, IH), 12.36 (bs, IH); 13C NMR: 55.25, 101.10, 108.77, 111.17, 111.24, 111.51, 112.31, 121.45, 122.26, 122.43, 122.89, 123.21, 125.73, 126.27, 131.75, 138.75, 153.44, 168.50; El m/z (%): 306 (4.1), 276 (34), 205 (100) ; HRMS:
C18H14N203 requires 306.1004, found 306.0999.

3-(5fluoro-indol-3 yl)-indole-5-carboxylic acid (Of) = AcOH

[00312] Purification by flash chromatography using 2:1 hexanes:THF, 5%AcOH. Yellow powder (0.324 g, 61%); mp 225 C (dec.); tH NMR: 1.91 (s, 3H), 7.01 (dt, 1H, J=9.0(t), J=2.5), 7.48 (m, 3H), 7.71 (d, 1H, J=2.4), 7.75 (d, 1H, J=2.2), 7.78 (dd, IH, J=8.5, J=1.5), 8.40 (s, 1H), 11.32 (s, IH), 11.55 (s, IH), 12.21 (bs, 2H);

"C NMR: 21.08, 103.87, 104.06, 109.25, 109.29, 109.39, 109.60, 110.50, 111.30, 112.57, 112.65, 121.54, 122.00, 122.54, 123.49, 124.28, 125.59, 126.08, 126.15, 133.05, 138.76, 156.20, 158.04, 168.45, 171.99; El m/z (0/6): 294 (0.5), 60 (100);
HRMS: C17H11FNZ02 requires 294.0804, found 294.0801.

3-(5-chloro-indol-3 yl)-indole-S-carboxylic acid (0g) = AcOH

[00313] Purification by flash chromatography using 2:1 hexanes:THF, 5%AcOH. Beige solid (0.328 g, 59%); mp 270 C (dec.);'H NMR: 1.91 (s, 3H), 7.16 (dd, 1H, J=8.6, J=2.0), 7.49 (d, IH, J=8.6), 7.51 (d, IH, J=8.5), 7.75 (m, 4H), 8.39 (s, IH), 11.43 (s, IH), 11.57 (s, 1H), 12.21 (bs, 2H); 13C NMR: 21.05, 108.83, 110.18, 111.34, 113.20, 118.42, 121.31, 121.56, 121.93, 122.57, 123.69, 123.75, 124.04, 125.62, 127.09, 134.83, 138.76, 168.42, 171.98; El m/z (%): 310 (1.0), 205 (88), 60 (100); HRMS: C17H>>C1N202 requires 310.0509, found 310.0506.
3-(5-methyl-indol-3-yl)-indole-5-carboxylic acid (0h) = AcOH

[00314] Purification by flash chromatography using 2.6:1 hexanes:THF, 5%
AcOH. Beige powder (0.159 g, 45%); mp 215 C; 'H NMR: 1.91 (s, 3H), 2.41 (s, 3H), 6.98 (d, 1H, J=7.9), 7,36 (d, 1H, J=8.2), 7.50 (d, 1H, J=8.5), 7.54 (s, 1H), 7.56 (d, 1H, J=2.1), 7.71 (d, 1 H, J=2.0), 7.77 (dd, 1 H, J=8.5, J=1.1), 8.41 (s, 1 H), 11.06 (s, IH), 11.50 (s, 1H), 12.22 (bs, 2H); 13C NMR: 21.10, 21.33, 108.42, 111.18, 111.23, 111.32, 118.97, 121.46, 122.18, 122.23, 122.46, 122.93, 123.30, 125.78, 126.27, 127.38, 134.75, 138.73, 168.51, 172.01; El m/z (%): 290 (100), 220 (27), 205 (83);
HRMS: C18H14N202 requires 290.1055, found 290.1055.

3-(5-(trifluoromethoxy)-indol-3-yl)-indole-5-carboxylic acid (0i) = AcOH

[00315] Purification by flash chromatography, run twice, using 2:1 hexanes:THF, 5% AcOH. Yellow powder (0.137 g, 33%); mp 220 C (dec.); 'H
NMR: 1.91 (s, 3H), 7.14 (dd, 1H, J=8.7, J=1.1), 7.51 (d, 1H, J=8.5), 7.56 (d, IH, J=8.8), 7.63 (s, 1H), 7.78 (m, 3H), 8.39 (s, 1H), 11.52 (s, 1H), 11.57 (s, 1H), 12.25 (bs, 2H); 13C NMR: 21.08, 109.58, 110.11, 111.36, 111.61, 112.74, 114.98, 117.45, 119.47, 121.49, 121.69, 121.92, 122.61, 123.52, 123.70, 124.69, 125.60, 126.05, 134.86, 138.77, 141.89, 168.43, 172.00; El m/z (0/6): 360 (5.5), 220 (19), 205 (38), 60 (100); HRMS: C18H11F3N203 requires 360.0721, found 360.0721.
3-(5-Hydroxy-indol-3-yl)-indole-5-carboxylic acid (0j) [00316] Purification by flash chromatography using 1:1 hexanes:EtOAc, 5%
AcOH. Brown solid (0.082 g, 28%); mp 85-88 C; 'H NMR S: 6.67 (dd, IH, J=8.6, J=2.3), 7.04 (d, 1H, J=2.3), 7.25 (d, IH, J=8.6), 7.48 (m, 2H), 7.57 (d, 1H, J=2.2), 7.76 (dd, 1H, J=8.5, J=1.4), 8.38 (s, IH), 8.64 (bs, 1H), 10.89 (d, 1H, J=1.8), 11.44 (s, 1H), 12.28 (bs, 1H); 13C NMR 5: 103.24, 108.12, 111.08, 111.41, 111.62, 111.90, 122.15, 122.48, 122.64, 122.80, 125.76, 126.82, 130.89, 138.56, 150.76, 168.94; ESI
m/z (%): 291.1 [M-1 ] (100).

3-(Indol-3 yl)-indole-6-carboxylic acid (0k) = AcOH

[00317] Purification by flash chromatography using 2:1 hexanes:EtOAc, 5%
AcOH. Yellow solid (0.155 g, 37%); mp 205 C (dec.); 1 H NMR S: 1.91 (s, 3H), 7.07 (t, 1 H, J=7.1), 7.15 (t, 1 H, J=7.2), 7.45 (d, 1 H, J=8.0), 7.68 (m, 2H), 7.78 (d, 1 H, J=7.9), 7.83 (d, 1 H, J=8.4), 7.88 (d, 1 H, J=2.4), 8.11 (s, 1 H), 11.21 (s, I
H), 11.54 (s, 1H), 12.30 (bs, 2H); 13C NMR 5: 21.08, 109.02, 110.26, 111.59, 113.65, 118.89, 119.13, 119.44, 119.77, 121.25, 122.14, 123.53, 125.42, 125.92, 129.08, 135.64, 136.35, 168.41, 172.00; El m/z (1/6): 276 (3.5), 232 (100); HRMS: C17H12N202 calculated 276.0899, found 276.0901.

3-(Indol-3 yl)-indole-7-carboxylic acid (01) [00318] Suspended and sonicated solid in EtOAc (5 mL), filtered, rinsed with EtOAc. Yellow solid (0.349 g, 63%); mp 245 C (dec.); 'H NMR: 7.07 (t, 1H, J=7.5), 7.18 (m, 2H), 7.46 (d, IH, J=8.0), 7.62 (d, 1H, J=1.8), 7.67 (d, 1H, J=2.0), 7.71 (d, 1 H, J=7.9), 7.82 (d, 1 H, J=7.4), 8.04 (d, I H, J=7.8), 11.09 (s, 1 H), 11.22 (s, 1 H), 13.10 (bs, 1H); 13C NMR: 108.87, 110.07, 111.62, 113.98, 118.35, 118.93, 119.19, 121.26, 122.26, 123.08, 124.10, 124.88, 126.00, 127.68, 135.11, 136.37, 168.18; El m/z (%): 275.9 (100), 257.9 (28), 230.0 (31); HRMS: C17H12N202 calculated 276.0899, found 276.0902 (v) Cleavage of methoxyl groups to hydroxyl groups (0m) Me0 I j N I I N OMe 1) BBr3, DCM, -78 C to RT HO I N 11 N OH
H H H H
Ob 2) H20 Om Scheme 3, Conversion of Ob to Om 3-(5-hydroxy-indol-3 yl)-indol-5-ol (0m) [00319] The product Om was obtained by stirring Ob (0.923 g, 3.16 mmol) in dichloromethane (20 mL) at -78 C, to which was added BBr3 (3 mL, 10 eq.). The dark red solution was allowed to slowly warm to room temperature and stirred for 20 h. The reaction mixture was then cooled to 0 C, water added (10 mL), and the pH
raised to about 7 by adding 1N NaOH. The aqueous layer was extracted with EtOAc (2 X 50 mL) and the organic phase dried and concentrated, affording crude product.
The product was purified by flash column chromatography, using 1:1 hexanes:EtOAc with 5% MeOH as the eluent. Yellow-green solid (0.546 g, 65%); mp 235 C
(dec.);
'H NMR S: 6.64 (dd, 2H, J=8.6, ,T=2.3), 7.03 (d, 2H, J=8.3), 7.21 (d, 2H, J=8.6), 7.36 (d, 2H, J=8.4), 8.57 (s, 2H), 10.76 (d, 2H, J=1.5); 13C NMR 8: 103.99, 109.69, 111.84, 112.17, 122.38, 127.30, 131.29, 150.96.

Synthesis of One Carbon-Linked Bis-Indoles [00320] The one carbon-linked bis-indoles (la-g) were synthesized from indole or 5-substituted indole and formaldehyde, using the method of Jackson et al.
(J.

Chem. Soc. Perkin Trans. I, 1987, 11: 2543-51 (Scheme 4).

R R' H )~ H
I~ I + I~ I 1 a-g H H AcOH, H20 50-90 C, 3-20 hrs.
R R' H -OH -Scheme 4 Synthesis of 1C bis-indoles. la and lb produced as a mixture, as were lc and ld.

[00321] Formaldahyde (0.55 eq., 37% aqueous solution) and acetic acid (0.5 eq.) were added to indole or substituted indole (1.0 - 100 mmol) suspended in and the suspension heated at 90 C for 8-20 hrs. (Scheme 4). Since 5-hydroxy-indole is sensitive to oxidation, the synthesis of lg was performed under argon and excluding light, and was heated at only 50 C for 3 hrs. For the reaction giving la,b, as well as that for lc,d, the beige, gummy mixture was collected by vacuum filtration, washed with water, recrystallized from EtOAc/hexanes and the resulting cream solid purified by flash chromatography using 4:1 hexanes/EtOAc as eluant. Purification of le-g are given below.

Di-(indol-3 yl)methane (la):

[00322] White crystals (5.55g, 45%); mp 158-160 C;1H NMR: 4.13 (s, 2H), 6.91 (t, 2H, J=7.3), 7.03 (t, 2H, J=7.3), 7.13 (s, 2H), 7.31 (d, 2H, J=8.2), 7.51 (d, 2H, J=7.6), 10.72 (s, 2H); 13C NMR: 20.94, 111.32 (2C), 114.21 (2C), 118.04 (2C), 118.69 (2C), 120.76 (2C), 122.77 (2C), 127.21 (2C), 136.41 (2C); El m/z (%):
246.4 (6), 117.8 (100), 89.6 (49); HRMS: C H14N2 requires 246.1157, found 246.1147.
3-((indol-1 yl)methyl)-indole (lb):

[00323] White crystals (0.262g, 2.1%); mp 82-84 C; 'H NMR: 5.50 (s, 2H), 6.38 (d, 1H, J=0.6), 7.02 (m, 4H), 7.34 (d, 1H, J=8.2), 7.49 (m, 4H), 7.64 (d, IH, J=8.2), 11.03 (s, 1H); 13C NMR: 41.29, 100.30, 110.27, 111.02, 111.61, 118.56, 118.80 (2C), 120.38, 120.84, 121.29, 124.84, 126.50, 128.30, 128.84, 135.66, 136.36;
El m/z (%): 246.4 (8), 117.6 (100); HRMS: C HI4NZ requires 246.1157, found 246.1168.

Bis(5-j nethoxy-indol-3 yl)methane (lc):

[003241] Light grey powder (0.251g, 45%); mp 167-169 C; 'H NMR: 3.71 (s, 6H), 4.07 (s, 2H), 6.70 (dd, 2H, J=8.8, J=2.4), 7.03 (d, 2H, J=2.4), 7.09 (d, 2H, J=2.4), 7.22 (d, 2H, J=8.5), 10.56 (s, 2H); 13C NMR: 20.90, 55.34 (2C), 100.72 (2C), 110.75 (2C), ~ 11.93 (2C), 113.97 (2C), 123.50 (2C), 127.54 (2C), 131.58 (2C), 152.80 (2C);
EI m/;: (%): 306.4 (14), 147.6 (82), 104.6 (100); HRMS: C19H18N202 requires 306.1368, found 306.1358 .

5-met~oxy-3-((5-methoxy-indol-1 yl)methyl)-indole (ld):

[00325] Yellow crystals (0.062g, 11%); mp 48-50 C; 'H NMR: 3.65 (s, 3H), 3.72 (s, 3H), 5.43 (s, 2H), 6.31 (dd, 1H, J=3.1, J=0.6), 6.73 (m, 2H), 6.96 (d, 1H, J=2.4 ), 7.01 (d, 1H, J=2.1), 7.24 (d, 1H, J=8.8), 7.40 (d, 1H, J=2.5), 7.46 (d, 1H, J=3.1), 7.53 (d, 1H, J=8.8), 10.86 (s, 1H);13C NMR: 41.47, 55.31 (2C), 99.90, 100.69, 102.11, 110.92, 110.94, 111.15, 112.20, 125.24, 126.73, 128.69 (2C), 129.29, 131.06, 131.47, 153.35, 153.18; EI m/z (%): 306.4 (79), 160.5 (92), 147.5 (100);
HRMS: C19H18N202 requires 306.1368, found 306.1370.

3-((irdol-3 yl)methyl)-indole-S-carboxylic acid (le) [0031.61 Purification by flash chromatography in 1.5:1 hexanes:THF, 5%
AcOli, giving a light pink solid (0.045 g, 16%); mp 236-238 C; 'H NMR: 4.16 (s, 2H), 5.91 (t, IH, J=7.5), 7.02 (t, 1H, J=7.5), 7.10 (d, IH, J=2.2), 7.25 (d, 1H, J=2.0), 7.32 .;d, IH, J=8.1), 7.36 (d, 1H, J=8.3), 7.50 (d, 1H, J=8.0), 7.66 (dd, 1H, J=8.5, J=1.E), 8.19 (d, 1H, J=0.8), 10.74 (s, 1H), 11.10 (s, 1H), 12.25 (bs, 1H); 13C
NMR:
20.7S,, 110.96, 111.30, 113.82, 115.57, 118.03, 118.57, 120.77, 121.39, 122.12, 122.70, 124.39, 126.68, 127.06, 136.38, 138.85, 168.47; El m/z (0/0): 290 (5.8), 246 (91), 161 (100); HRMS: C18H14N2O2 requires 290.1055, found 290.1053.
Bis-(indole-5-carboxylic acid-3-yl)methane (lf) [00327] Recrystallized from THF/HZO, collected solid and purified filtrate by column chromatography in 1.5:1 hexanes:THF, 5% AcOH. White solid (0.152 g, 91%); mp 260 C (dec.); 'H NMR: 4.23 (s, 2H), 7.24 (d, 2H, J=2.0), 7.39 (d, 2H, J=8.6), 7.69 (dd, 2H, J=8.6, J=1.6), 8.19 (s, 2H), 11.14 (d, 2H, J=1.4), 12.31 (s, 2H);
13C NMR: 20.72, 111.07, 115.26, 120.67, 121.41, 122.19, 124.45, 126.87, 138.93, 168.37; El m/z (%): 335 (M+1, 0.04), 161 (100), 144 (61) ; HRMS: C19H14N204 requires 334.0953, found 334.0933.

Bis-(5-hydroxy-indol-3-yl)methane (1g) [00328] Purification by flash chromatography in 1:1 hexanes:EtOAc, 5%
AcOH. Light pink solid (0.248 g, 50%); mp 57-59 C;'H NMR: 3.92 (s, 2H), 6.55 (dd, 214, J=8.6, J=2.3), 6.78 (d, 2H, J=2.3), 6.95 (d, 2H, J=2.2), 7.09 (d, 2H, J=8.6), 8.47 (s, 2H), 10.38 (d, 2H, J=1.3); 13C NMR: 21.14, 102.69, 110.98, 111.45, 113.09, 123.13, 127.84, 130.95, 149.84; El m/z (%): 279 (M+1, 1.0), 74 (75), 59 (100);
HRMS: C17H14N2O2 requires 278.1055, found 278.1054.

[00329] In addition to the methylene-linked bis-indoles, three bis-(5-hydroxyindole) compounds (1h,i,j) were generated which also had the aromatic groups separated by one carbon. These were obtained by condensing 5-hydroxyindole with one, two or three units of acetone in the presence of trifluoroacetic acid (Scheme 5), following the procedure used for unsubstituted indole.

The one-acetone condensation product lh was produced within seconds at room temperature, with the two- and three-acetone products (li,j) becoming the dominant species after several hours and several days, respectively.

Ho ~ ~ TFA Time Major species (TLC) Oto - 6hrs lh H RT 6 hrs. to 3-5 days 1 i > 5 days 1 j Scheme 5. Condensation of 5-hydroxyindole with acetone to give lh,i,j.

[00330] The highly branched bridging groups of lh,i,j provide additional molecular diversity to help identify favourable characteristics for compound activity against A(3. Furthermore, these compounds are expected to be more stable than the methylene-linked species la-g, which are likely easily oxidized at the CH2 bridge.
The observation that lh,i,j remained a light colour when exposed to light and air, while la-g become darkly coloured within a few days or weeks, supports this notion.
Condensation of 5-hydroxyindole with acetone (lh,i,j):

[00331] 5-Hydroxyindole (0.133 g, 1.00 mmol) was stirred at room temperature in acetone (2 mL) and TFA (0.150 mL, 2.0 eq.). Upon completion (5 min. for lh, or 18 h for a mixture of 1i and lj), the reaction mixture was neutralized with saturated NaHCO3(aq.), and the product(s) extracted with EtOAc (2 x 20 mL). Products were purified by flash column chromatography in 1.5:1 (lh) or 2:1 (li,j) hexanes:EtOAc, both containing 5% MeOH.

2,2-Bis-(5-hydroxyindol-3 yl)propane (lh) [00332] Light beige solid (0.099 g, 65%); mp 133-136 C;'H NMR S: 1.72 (s, 6H), 6.45 (dd, 2H, J=8.6, J=2.3), 6.53 (d, 2H, J=2.3), 7.05 (d, 2H, J=8.6), 7.07 (d, 2H, J=2.5), 8.27 (s, 2H), 10.38 (d, 2H, J=2.0);13C NMR 6: 29.67, 33.91, 104.64, 110.58, 111.32, 121.17, 122.71, 126.57, 131.65, 149.04; EI m/z (%): 306.0 (3.8), 133 (12) 59 (100); HRMS: C19H18N202 calculated 306.1368, found 306.1374.

1,2,3,4-Tetrahydro-3-(5-hydroxyindol-3 yl)-1,1,3-trimethylcyclopenta[bJindol-7-ol (1i) [00333] White solid (0.126 g, 73%); mp 132 C (dec.); 'H NMR 5: 1.35 (s, 3H), 1.42 (s, 3H), 1.71 (s, 3H), 2.36 (d, IH, J=12.7), 2.75 (d, 1H, J=12.7), 6.48 (dd, IH, J=8.6, J=2.6), 6.54 (dd, 1H, J=8.6, J=2.3), 6.56 (d, 1H, J=2.0), 6.79 (d, 1H, J=2.2), 6.91 (d, 1 H, J=2.5), 7.00 (d, 1 H, J=8.6), 7.10 (d, 1 H, J=8.5), 8.45 (s, 2H), 10.28 (s, IH), 10.45 (d, 111, J=1.9);13C NMR 6: 28.72, 30.04, 30.58, 38.13, 41.66, 61.81, 102.18, 103.73, 109.17, 110.96, 111.64, 111.92, 121.27, 121.76, 122.89, 123.60, 125.93, 131.58, 135.35, 148.70, 149.65, 150.05; EI m/z (%): 346 (22), 331 (74), 56 (100); HRMS: C22H22N202 calculated 346.1681, found 346.1690.

3,3,3 ;3'-Bis(1,2,3,4-tetrahydro-l,l-dimethylcyclopenta[bJindol-7-ol) (lj) [00334] White solid (0.042 g, 22%); mp 140 C (dec.); 'H NMR S(acetone-d6):
1.47 (s, 6H), 1.53 (s, 6H), 2.58 (d, 2H, J=12.9), 2.80 (d, 2H, J=12.9), 6.65 (dd, 2H, J=8.6, J=2.3), 6.95 (d, 2H, J=2.0), 7.12 (d, 2H, J=8.6), 7.54 (s, 2H), 9.50 (s, 2H);13C
NMR S(acetone-d6): 31.05, 31.58, 40.39, 50.89, 63.97, 104.2, 111.5, 113.6, 125.89, 127.43, 138.15, 147.82, 152.30; El m/z (%):386 (14),198 (64), 174 (100); HRMS:
C25H26N202 calculated 386.1994, found 386.1995.

Synthesis of Two Carbon-Linked Bis-Indoles [003351 Directly linked bis-indoles were synthesized according to the procedure set forth in Scheme 6 below:

O O
Br 1) EtMgBr, Et20, -78 C Br. Br (/ I NI I
N
H 2) C1~O /CI,-20 C-r RT H H
~f BH3, THF
RT, 8 hrs.
1) KH, THF, O C
2) f-BuLi, -78 C Br Br 2b +2c ~ ( \ I ~ I
3) C02(), ffN -78 C N ~
H H
SCHEME 6, Synthesis of ethyl-linked bis-indoles 2b and 2c (i) Formation of 3,3'-oxalyl-linked bis-(5-bromo-indole) [00336] A suspension of 5-bromo-indole (3.92 g, 20 mmol) in Et20 (100 mL) was cooled to -20 C and ethylmagnesium bromide (7.3 mL, 3.0 M in THF, 1.1 eq.) added dropwise. The beige suspension was warmed to room temperature for 3 hrs.
before again cooling to -20 C and adding oxalyl chloride (0.87 mL, 0.50 eq.) dropwise, causing an oily orange/brown precipitate to form. After stirring for 3 hrs., water (50 mL) was added and the suspension sonicated for 30 min. A yellow solid was collected by vacuum filtration, suspended in MeOH (30 mL) and stirred overnight to dissolve impurities before again being collected by vacuum filtration.

1, 2-bis(5-bromo-indol-3-yl)ethane-1,2-dione [00337] Yellow solid (0.900 g, 20%); mp N.D.; 'H NMR: 7.44 (dd, 2H, J=8.5, J=2.0), 7.35 (d, 2H, J=8.5), 8.35 (d, 2H, J=3.5), 8.43 (d, 2H, J=1.5), 12.42 (s, 2H); 13C
NMR: 107.60, 112.44, 115.30, 115.88, 124.11, 126.72, 128.11, 136.13, 139.22, 187.60; ; El m/z (%): 446 (13), 232 (100), 222 (39); HRMS: C,BH~oBrzN2O2 requires 443.9109, found 443.9114.

(ii) Reduction to ethyl-linked bis-(5-bromo-indole) [00338] To a solution of 1,2-bis(5-bromo-indol-3-yl)ethane-1,2-dione (0.900 g, 2.03 mmol) in THF (20 mL) was added BH3=THF (10.8 mL, 1.0 M in THF, 5 eq.).
The solution was stirred at room temperature for 8 hrs. before slowly quenching the reaction with MeOH (20 mL). The solvent and the B(OMe)3 formed were removed under vacuum and the remaining solid sonicated with MeOH (10 mL). The MeOH
and any remaining B(OMe)3 were removed and the solid purified by flash chromatography using 3.5:1 CHC13:hexanes as the solvent system. The product, 1,2-bis(5-bromo-indol-3-yl)ethane, was obtained as a white powder and used without further purification.

(iii) Carboxylation to 2b and 2c [00339] The carboxylation of 1,2-bis(5-bromo-indol-3-yl)ethane to 2b and 2c was carried out as described in Example 1, paragraph iv. After adding COZ(s), the reaction was quenched with MeOH (5 mL) and H20 (20 mL), and the basic solution extracted twice with EtOAc (20 mL) before acidifying to pH 3 with 1 N HC1. TLC
suggested that the resulting white precipitate was primarily a mixture of 2b and 2c (i.e. the mono- and dicarboxylated species). These were separated by flash chromatography in 2:1 hexanes:THF, 5% AcOH to isolate 2b, followed by 1:1 hexanes:THF, 5% AcOH, to isolate 2c. Purification of 2b was achieved by recrystallizing from THF/hexanes, while 2c was purified by refluxing in EtOH
(10 mL) for 30 min. before being collected by vacuum filtration.
3-(2-(5-bromo-indol-3-yl)ethyl)-indole-5-carboxylic acid (2b) [00340] Light yellow powder (0.062 g, 16%); mp 205 C (dec.); 'H NMR: 3.07 (m, 4H), 7.16 (dd, 1H, J=8.6, J=1.8), 7.24 (m, 2H), 7.30 (d, 1H, J=8.6), 7.36 (d, 1H, J=8.5), 7.67 (d, 1H, J=1.7), 7.72 (d, 1H, J=8.5), 8.25 (s, 1H), 11.03 (s, 1H), 11.09 (s, 1H);13C NMR: 25.41, 25.44, 110.73, 110.77, 113.28, 114.46, 115.89, 120.57, 120.79, 122.30, 123.18, 123.65, 124.00, 126.66, 129.06, 134.85, 138.40, 169.06; El m/z (%):
382 (0.5), 338 (8.0), 130 (100); HRMS: C19H15BrNZO2 requires 382.0317, found 382.0314.

1,2-bis-(indole-5-carboxylic acid-3-yl)ethane (2c) [00341] White powder (0.031 g, 9%); mp 255 C (dec.); 'H NMR: 3.13 (s, 411), 7.29 (d, 2H, J=2.1), 7.40 (d, 2H, J=8.5), 7.71 (dd, 2H, J=1.3), 8.25 (s, 2H), 11.15 (s, 2H), 12.30 (s, 2H);13C NMR: 25.33, 111.02, 116.02, 120.73, 121.01, 122.18, 123.90, 126.73, 138.70, 168.45.

Synthesis of 3-(7-azaindol-3-yl)-indoles R O
OH
H O EtOH, 45 -> 25 C R BH3 -THF
+ H O H N --Piperidine (cat.) THF
I I g-24 hrs. 9n, R = OMe 12-20 hrs.
N H 9o, R = Br R ( I ~ I I~ H~ R I~ I I I, H H ~I H N

10n, R = OMe 10p, R OMe 10o, R=Br 10q, RBr ~
1) BBr3, DCM, -78 C to RT HO
10n 1 ~~
2) H2O H N
On 1) KH, THF, O C
HOZC H+ HOzC
100 2) t-BuLi, -781C I/
N N N
3) CO2(s), -78 C H BH3 H H

Oo Oq 1) BBr3, DCM, -7B C to RT HO
10p - k I ~~
2) H20 H H N
Op Scheme 7, Synthesis of 7-azaindole- and 2,3-dihydro-7-azaindole-containing compounds.

3-(5-methoxyindol-3 yl)-7-azaindole - BHj (10n) [00342] The procedure from Example 1, paragraph (i) was used to give 9n (Scheme 6), a light orange solid that was rinsed with EtOH (5 mL) and dried under vacuum (2.52 g, 85%). The procedure from Example 1, paragraph (ii) was used to give 10n from 9n. Purification of lOn was achieved by flash column chromatography using 1.7:1 hexanes:EtOAc as the eluent. Finally, the product was recrystallized from EtOH, giving yellow crystals (1.22 g, 58%). 1H NMR (DMSO-d6): 2.58 (bs, 3H), 3.78 (s, 3H), 6.83 (dd, 1H, J=8.8, J=2.4), 7.16 (d, 1H, J=2.3), 7.35 (m, 2H), 7.72 (d, 1H, J=2.5), 7.78 (d, 1H, J=2.4), 8.32 (d, 1H, J=5.5), 8.51 (d, 1H, J=7.8), 11.22 (s, 1H), 11.94 (s, 1H); 13C NMR: 55.86, 56.51, 101.41, 107.53, 111.21, 112.94, 115.79, 122.67, 123.97, 124.30, 126.51, 132.03, 132.77, 141.27, 143.06, 154.25.
3-(5-bromoindol-3 yl)-7-azaindole - BH3 (l0o) [00343] Same procedure as for lOn (Scheme 6). Purification after the coupling step was accomplished by flash column chromatography using 2:1 EtOAc:hexanes, 5% MeOH as the eluent, giving a yellow-orange solid (2.40 g, 97%).
Purification of l0o after BH3 reduction was accomplished by flash column chromatography using 1.2:1 hexanes:EtOAc, giving a bright yellow solid (1.13 g, 54%). 'H NMR (DMSO-db): 2.58 (bs, 3H), 7.29 (dd, IH, J=8.6, J=1.8), 7.36 (dd, IH, J=7.9, J=5.6), 7.45 (d, 1H, J=8.6), 7.85 (m, 3H), 8.33 (d, IH, J=5.5), 8.52 (d, 1H, J=7.9), 11.60 (s, 1H), 12.02 (s, 1H); 13C NMR: 107.52, 110.22, 112.47, 114.24, 115.91, 121.70, 122.48, 124.45, 124.60, 125.23, 127.90, 132.64, 135.54, 141.37, 143.01.

3-(5-methoxyindol-3 yl)-7-azaindole (lOp) [00344] To remove the BH3 group, crude lOn (approx. 1.5 g, 6 mmol) was sonicated in a mixture of AcOH and 1N HCl (1:1, 50 mL) until TLC indicated completion (1 hr.). The reaction was neutralized with saturated NazCO3 (aq.) until the pH reached 7-8, and extracted with EtOAc (2 X 20 mL). The organic phase was dried (Na2SO4) and concentrated, and the product (10n) purified by recrystallization from THF/hexanes. Further product was obtained from the filtrate after purifying by column chromatography using 1:1 hexanes:THF as eluent. Yellow solid, 1.45 g (approx. 100 %).1H NMR (DMSO-d6): 3.34 (s, 3H), 6.80 (dd, 1H, J=8.8, J=2.3), 7.11 (dd, 1 H, J=7.8, J=4.6), 7.21 (d, 1 H, J=2.0), 7.3 3 (d, 1 H, J=8.7), 7.64 (d, 1 H, J=2.3), 7.76 (d, 1 H, J=2.2), 8.16 (d, 1 H, J=7.8), 8.26 (d, 1 H, J=4.6), 11.07 (s, 1 H), 11.68 (s, 1H); 13C NMR: 55.32, 101.17, 108.77, 108.78, 111.47, 112.23, 115.25, 118.20, 121.78, 122.84, 126.01, 127.69, 131.50, 142.64, 148.69, 153.49.
3-(2,3-dihydro-7-azaindol-3 yl)-indol-S-ol (On) [003451 Conversion of lOn to On was carried out using the procedure from Example 1, paragraph (v). In the course of the reaction, however, the 2,3 double bond of 7-azaindole was reduced during the reaction by the BH3 present. The product was purified by chromatography using 12:1 CHC13:MeOH as the eluent, affording On as a light yellow solid (0.610 g, 61%). 'H NMR (DMSO-d6): 3.41 (t, IH, J=9.1), 3.73 (t, 1H, J=9.1), 4.57 (t, 1H, J=9.2), 5.08 (s, 1H), 6.29 (d, 1H, J=1.9), 6.39 (d, 1H, J=2.4), 6.41 (d, 1H, J=2.3), 6.96 (dd, 1H, J=7.8, J=4.7), 7.30 (s, 1H), 7.76 (d, 1H, J=7.6), 8.17 (dd, 1H, J=4.6, J=1.2), 8.31 (d, 1H, J=4.1), 11.39 (s, IH); 13C NMR: 40.44, 55.14, 110.37, 112.21, 113.99, 115.27, 115.72, 118.91, 123.28, 127.73, 133.76, 142.93, 144.92, 149.47, 150.20.

3-(7-azaindol-3-yl)-indole-5-carboxylic acid = BH3 (0o) [00346] Carboxylation of IOo to Oc followed the same procedure as for Oc-I
(Example 1, paragraph (iv)). Purification of the product was achieved by recrystallization from EtOH/HzO, giving a yellow solid (0.383 g, 45 %). 'H NMR

(DMSO-d6): 2.58 (bs, 3H), 7.38 (dd, IH, J=7.9, J=5.6), 7.53 (d, 1H, J=8.6), 7.82 (m, 2H), 7.93 (d, IH, J=2.4), 8.36 (d, 1H, J=5.5), 8.43 (s, IH), 8.56 (d, 1H, J=7.8), 11.77 (d, 1H, J=1.5), 12.07 (s, 1H), 12.53 (s, 1H); 13C NMR: 108.56, 109.88, 111.57, 115.51, 121.50, 121.92, 121.99, 122.85, 123.83, 124.69, 125.30, 132.27, 138.83, 141.00, 142.54, 168.29.

3-(7-azaindol-3-yl)-indole-5-carboxylic acid = AcOH (0p) [00347] Conversion of Oo to Op was the same as for 10p. Purification of the product was achieved by column chromatography, using 1.2:1 hexanes:THF, 2%
AcOH as the eluent, giving a yellow solid (0.125 g, approx. 100 %). 'H NMR
(DMSO-d6): 1.92 (s, 311), 7.14 (dd, 1H, J=7.9, J=4.6), 7.51 (d, 1H, J=8.6), 7.74 (d, 1H, J=2.4), 7.78 (m, 2H), 8.16 (dd, 1H, J=7.9, J=1.2), 8.29 (dd, 1H, J=4.6, J=1.4), 8.42 (d, IH, J=0.6), 11.61 (s, 1H), 11.79 (s, 1H), 12.20 (bs, 2H) ; 13C NMR:
21.03, 107.94, 110.28, 111.37, 115.47, 118.21, 121.59, 121.99, 122.30, 122.61, 123.75, 124.88, 125.48, 138,79, 142.87, 148.67, 168.39, 171.97.

3-(7-azaindol-3 yl)-indol-5-ol (0p) [00348] Conversion of lOp to Op was carried out using the procedure from Example 1, paragraph (v). The product was purified by chromatography using 8:1 CHC13:MeOH as the eluent, affording Op as a yellow solid (0.483 g, 65%). 'H
NMR
(DMSO-d6): 6.68 (dd, 1H, J=8.6, J=2.2), 7.10 (m, 211), 7.23 (d, IH, J=8.6), 7.56 (d, IH, J=2.4), 7.60 (d, 1H, J=2.3), 8.14 (d, 1H, 1=7.8), 8.25 (d, 1H, J=4.6), 8.65 (s, IH), 10.90 (s, 1H), 11.62 (s, 1H); 13C NMR: 103.31, 108.08, 109.15, 111.60, 111.90, 115.19, 118.21, 121.35, 122.60, 126.49, 127.79, 130.89, 142.59, 148.59, 150.79, Synthesis of Indole-Phenyl Compounds HOZC
HO2C 1) EtMgBr, THF, 0 C to RT N R OMe H
H 2) Me0 Br 100,R + 101,R=H
2.2 eq. (for 100) or 1.Oeq.(for101) OMe 1) BBr3, DCM, -78 C to RT HO2C

2) HZO N I R OH
H
102, R= + 103, R= H

OH
Scheme 8: Synthesis of indole-phenyl compounds 100-103.
2,3-bis(4-methoxybenzyl)-indole-5-carboxylic acid (100) [00349] To solution of indole-5-carboxylic acid (0.322 g, 2.0 mmol) in THF
(20 mL) at 0 C was added EtMgBr (2.2 eq., 3.0 M solution in THF), giving a gummy white suspension. 1-(Bromoethyl)-4-methoxybenzene (2.2 eq.) was added dropwise and the suspension warmed to room temperature and stirred overnight. Water was added (10 mL), followed by I N HC1(aq) until the pH was 2. The reaction mixture was extracted with EtOAc (20 mL x 2) and the organic phase dried with MgSO4.
Purification of 101 was achieved by column chromatography using 4:1 hexanes:EtOAc,3% AcOH as the eluent, followed by recrystallization from EtOH/HZO. The product was obtained as a white solid (0.345 g, 43%). 1H NMR
(DMSO-d6): 3.68 (s, 3H), 3.70 (s, 3H), 4.04 (s, 214), 4.05 (s, 2H), 6.78 (d, 2H, J=8.6), 6.83 (d, 2H, J=8.6), 7.06 (d, 2H, J=8.5), 7.13 (d, 2H, J=8.5), 7.30 (d, 1H, J=8.5), 7.62 (dd, 1H, J=8.5, J=1.2), 7.96 (s, 1H), 11.14 (s, IH), 12.22 (s, 1H);13C NMR:
28.93, 31.22, 55.43, 55.53, 110.85, 111.65, 114.13, 114.33, 121.24, 121.44, 122.35, 128.09, 129.46, 129.91, 131.75, 133.95, 137.41, 138.72, 157.81, 158.23, 168.89.
2,3-bis(4-hydroxybenzyl)-indole-5-carboxylic acid (102) 1003501 Conversion of 100 to 102 was carried out using the same procedure as for Om (Example 1, paragraph (v)). The product was purified by column chromatography using 1.3:1 hexanes:EtOAC, 5% AcOH as the eluent, giving a light pink solid (0.190 g, 85%). 1H NMR (DMSO-d6): 3.97 (s, 2H), 3.99 (s, 2H), 6.60 (d, 2H, J=8.2), 6.65 (d, 2H, J=8.2), 6.94 (d, 2H, J=8.3), 7.00 (d, 2H, J=8.3), 7.28 (d, 1H, J=8.5), 7.61 (d, 1H, J=8.5), 7.96 (s, 1H), 9.10 (bs, 211), 11.08 (s, 1H), 12.21 (bs, 1H);
"C NMR: 29.00, 31.27, 110.78, 111.78, 115.45, 115.62, 121.23, 121.27, 122.26, 128.16, 129.41, 129.84, 129.96, 132.18, 137.52, 138.67, 155.71, 156.19, 168.93.
3-(4-hydroxybenzyl)-indole-5-carboxylic acid (103) [003511 101 was synthesized in the same manner as 100, using only 1.0 eq. of 1-(bromoethyl)-4-methoxybenzene instead of 2.2 eq., and 103 was made from 101 using the same procedure as for 102. The product (103) was purified by column chromatography using 1.6:1 hexanes:EtOAc, 5% AcOH as the eluent, followed by recrystallization from EtOH/H2O, giving cream-colored needles (0.110 g, 33 %).
IH
NMR (DMSO-d6): 3.96 (s, 2H), 6.65 (d, 2H, J=8.4), 7.05 (d, 2H, J=8.4), 7.21 (d, 1H, J=1.8), 7.38 (d, 1H, J=8.5), 7.68 (dd, 1H, J=8.5, J=1.5), 8.09 (s, 1H), 9.11 (s, 1H), 11.16 (s, 1H), 12.31 (s, IH);13C NMR: 30.41, 111.55, 115.50, 116.45, 121.27, 121.84, 122.72, 125.11, 127.04, 129.65, 131.92, 139.42, 155.80, 168.87.

Suzuki Coupling of Various Bicyclic Aromatics (i) Synthesis of 3-(Benzofuran-2-yi)-indole-5-carboxylic acid (57) and 3-(Benzothiophen-2-yl)-indole-5-carboxylic acid (58) HOzC \ Br z HOzC Br CHaI MOOzC Br TsCI MeOzC WI~N
BrN DMF KZCOs,DMF NaOH 49 H 50 H 5i H CH2CI2 52 Ts Scheme 9. Preparation of 3-Bromo-l-(toluene-4-sulfonyl)-indole-5-carboxylic acid methyl ester (52) OH
BOH MeOzC KOHHOZC ~
53 ~ MeOH/H20 ~ Pd(OAc)z, K2C03 N 80 C ~ N
f f Br DMF, 90 C 54 Ts 57 H
MeOzC
~ \
~ N
52 Ts Pd(OAc)z, K2CO3 DMF, 90 C ~ ~
1OH MeOZC Z KOH ZI
B HO C
OH MeoHlH2O z 55 N 80 C N
H
56 Ts Scheme 10. Synthesis of 3-(Benzofuran-2-yl)-indole-5-carboxylic acid (57) and (Benzothiphene-2-y1)-indole-5-carboxylic acid (58) 3-Bromo-indole-5-carboxylic acid (50) [00352) To a solution of commercially available indole-5-carboxylic acid (1.0 g, 6.2 mmol) in DMF (10 mL) was added Br2 (334 L, 1.05 eq.) at room temperature (Scheme 9). Upon completion (-5 min), the reaction mixture was poured to an ice-cold solution of Na2SO3 (1 % in H20, 100 mL), resulting to the precipitation of the product, which was filtered and dried under high vacuum pressure. The isolated product was used without further purification. Light beige solid (1.46 g, 98%). 'H
NMR (DMSO): 7.47 (d, 1H, J=8.6), 7.65 (d, 1H, J=2.5), 7.76 (d, 1H, J=7.3, 1.3), 8.04 (s, 1H), 11.77 (s, IH), 12.57 (s, 1H).

3-Bromo-indole-5-carboxylic acid methyl ester (51) [00353] To a mixture of 50 (1.0 g, 4.2 mmol) and K2CO3 (0.865 g, 1.5 eq.) in DMF (15 mL) was added CH3I (337 L, 1.3 eq.) at room temperature (Scheme 9).
After stirring for 1.2 hours, the reaction was quenched with a saturated solution of NH4 Cl (10 mL). The aqueous layer was extracted with ethyl acetate (3 x 15 mL) and the combined organic layer was dried with MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (15%
ethyl acetate in hexane) to afford 51(1.01 g, 95%). 'H NMR (DMSO): 3.87 (s, 3H), 7.53 (d, IH, J=8.6), 7.71 (s, IH), 7.35 (dd, 1 H, J=8.6, 1.5), 7.64(s, 1 H), 11.41 (s, 1 H).
3-Bromo-l-(toluene-4-sulfonyl)-indole-5-carboxylic acid methyl ester (52) [00354] To a mixture of 51 (0.800 g, 3.15 mmol) and powdered NaOH (0.139 g, 1.1 eq.) in CH2ClZ (25 mL) was addedp-toleuenesulfonyl chloride (0.661 g, 1.1 eq.) at room temperature (Scheme 9). After stirring for 2.5 hours, the reaction was quenched with saturated solution of NH4Cl (20 mL). Layers were separated. The aqueous layer was extracted with CH2C12 (3 x 15 mL). The combined organic layer was dried with MgSO4, filtered and concentrated under reduced pressure.
Purification by flash column chromatography using 10% ethyl acetate in hexane as solvent system afforded 52 (1.081 g, 84%) as light orange solid. 'H NMR (DMSO): 2.34 (s, 3H), 3.89 (s, 3H), 7.43 (d, 2H, J=8.3), 7.97 (s, 1H), 7.98 (s, 1H) 8.03-8.05 (m, 2H), 8.15 (d, IH, J=9.14), 8.30 (s, IH).

Suzuki coupling of 3-bromo-l-(toluene-4-sulfonyl) -indole-5-carboxylic acid methyl ester (52) with benzofuran-2-boronic acid (53) to give 54 [00355] To a degassed solution of 52 (0.200 g, 0.489 mmol) was added Pd(OAc)2 (0.006 g, 0.05 eq.), K2C03 (0.135 g, 2 eq.) and benzofuran-2-boronic acid 53 (0.103 g, 1.3 eq.) at room temperature (Scheme 10). After degassing and purging with argon (repeated thrice), the reaction mixture was stirred at 90 C for 2.5 hours.
The mixture was allowed to cool to room temperature and diluted with H20 (15 mL).
The aqueous solution was extracted with ethyl acetate (5 x 15 mL) and combined organic layer was concentrated under reduced pressure. The residue was purified by flash column chromatography using 20 % ethyl acetate in hexane as solvent system to afford 54 (0.168 g, 77.1%) as white solid. 1H NMR (CDC13) 2.34 (s, 3H), 3.97 (s, 3H), 7.11 (s, 1 H), 7.23-7.27 (m, 3H), 7.31 (t, 1 H, J=7.3), 7.54 (d, 1 H, J=8.1), 7.61 (d, 1H, J=7.6), 7.84 (d, 2H, J=8.2), 8.09 (2s, 2H), 8.13 (s, 1H), 8.65 (s, 1H);

(CDC13) 21.64, 51.88, 103.27, 110.71, 113.63, 114.29, 120.95, 123.18, 124.65, 124.88, 126.08, 126.53, 127.05, 127.21, 128.88, 130.21, 134.74, 137.85, 145.76, 149.39, 154.30, 166.38 Suzuki coupling of 1-benzenesulfonyl-3-bromo-indole-5-carboxylic acid methyl ester (52) with benzothiophene-2-boronic acid (55) to give 56 [003561 To a degassed solution of 52 (0.200 g, 0.489 mmol) was added Pd(OAc)Z ( 0.006 g, 0.05 eq.), KZC03 (0.135 g, 2 eq.) and benzothiophene-2-boronic acid 55 (0.105 g, 1.2 eq.) at room temperature (Scheme 10). After degassing and purging with argon (repeated thrice), the reaction mixture was stirred at 90 C
for 12 hours. The mixture was allowed to cool to room temperature and diluted with (15 mL). The aqueous solution was extracted with ethyl acetate (5 x 15 mL) and combined organic layer was concentrated under reduced pressure. The residue was purified by flash column chromatography using 20 % ethyl acetate in hexane as solvent system to afford 56 (0.180 g, 82.7%) as white solid. 'H NMR (CDC13):
2.36 (s, 3H), 3.95 (s, 3H), 7.26 (d, 2H, J=8.5), 7.30-7.41 (m, 2H), 7.62 (s, 1H), 7.82-7.95 (m, 5H), 8.09 (s, 2H), 8.67 (s, 1H); 13C NMR (CDC13) 21.6, 52.3, 113.60, 117.88, 121.57, 122.16, 123.04, 123.63, 124.69, 124.75, 124.88, 126.06, 126.56, 127.05, 128.49, 130.22, 134.15, 134.77, 137.88, 138.97, 140.30, 145.75, 167.00.
Hydrolysis and De-tosylation of 54 and 56.

[003571 To a solution of 54 (0.336 mmol) or 56 (0.302 mmol) in MeOH/HZO
(7:1.25) was added KOH (7 eq.) (Scheme 10). The reaction mixture was stirred at 80 C overnight. The cooled solution was concentrated under reduced pressure and diluted with H20 (10 mL). The pH of the solution was adjusted to -2-3, resulting to the precipitation of the product. Finally, the indoles 57 and 58 were collected by vacuum filtration.

3-(Benzofuran-2-yl)-indole-5-carboxylic acid (57) [00358] Yellow solid (70.1 mg, 75.1%). 1 H NMR (CD3OD): 7.12 (s, 1H), 7.17-7.25 (m, 2H), 7.47-7.53 (m, 2H), 7.57 (d, 1H, J=6.9), 7.88 (s, 1H), 7.93 (dd, 2H, J=1.0, 8.6), 8.79 (s, 1H);13 C NMR (CD3OD): 98.94, 107.80, 110.02, 111.17, 119.86, 122.43, 122.67, 122.81, 122.95, 123.51, 124.00, 124.87, 130.04, 139.59, 152.80, 154.01, 170.90.

3-(Benzothiophene-2-yl)-indole-5-carboxylic acid (58) [00359] Light beige solid (63 mg, 72%). 1 H NMR (CD3OD): 7.25-7.37 (m, 2H), 7.50(d, 1H, J=8.6) 7.58 (s, IH), 7.73 (s, 1H), 7.78-7.84 (m, 2H), 7.92 (dd, 1H, J=1.4, 8.6), 8.793 (s, 1H).13C NMR (CD30D): 112.76, 113.49, 119.44, 123.0, 123.82, 123.98, 124.08, 124.84, 124.95, 125.61, 126.36, 126.54, 139.25, 139.82, 141.34, 142.54, 171.38.

(ii) Synthesis of 6-benzofuran-2-yl-naphthalen-2-ol (64), 6-benzothiophen-2-yl-naphthalen-2-ol (65), 6-(indol-3-yi]-naphthalen-2-ol (67) a) \ 1) t-Bul.i, THF
2) I2, THF I/ S I

b) \ 1) t-BuLi, THF ~ g 2) I2, THF I/ p I
c) I
C \ 1Z, KOH ~ \ TsCI, NaOH H DMF 61 H CHZCIZ

62 Ts Scheme 11. Preparation of 2-iodo-benzothiphene (59), 2-iodo-benzofuran (60) and 1-(toluene-4-sulfonyl)-3-iodo-indole (62) \ /
o Pd(OAC)2, K2C03 DMF
s ,OH I~~
'OH 59 HO 65 HO Pd(OAc)2, KZC03 Ts I
N
Pd(OAc)Z, K2C03 \ DMF

I / /
C'~-6N Ho 66 Ts KOH,MeOH

N
HO

Scheme 12. Synthesis of 6-Benzofuran-2-yl-naphthalen-2-ol (64), 6-Benzothiophen-2-yl-naphthalen-2-ol (65) and 6-[indol-3-yl]-naphthalen-2-ol (67) 2-iodo-benzothoiphene (59) [003601 To a solution of benzothiophene (1.0 g, 7.45 mmol) in THF (15 mL) was added t-BuLi (1.7 M, 4.8 mL, 1.1 eq.) at -78 C (Scheme 11). After stirring at the same temperature for 30 minutes, a solution of Iz (2.08 g, 1.1 eq.) in THF (5 mL) was added dropwise. The mixture was warmed up gradually to room temperature and stirred for 2 hours. The reaction was diluted with ethyl acetate (20 mL).
Excess 12 was reduced with 10% NazS2O7 solution. The organic layer was washed with brine (2 x mL), dried (MgSO4) and concentrated under reduced pressure. Purification by flash column chromatography using hexane afforded 59 (1.60 g, 82%) as white solid.
'H NMR (CDC13): 7.21-7.30 (m, 2H), 7.49 (s, IH), 7.7 (m, 2H).

2-iodo-benzofuran (60) [00361] To a solution of benzofuran (466 L, 4.2 mmol) in THF (15 mL) was added t-BuLi (1.7 M, 2.73 mL, 1.1 eq.) at -78 C (Scheme 11). After stirring at the same temperature for 30 minutes, a solution of IZ (1.I80 g, 1.1 eq.) in THF (5 mL) was added dropwise. The mixture was warmed up gradually to room temperature and stirred for 1.5 hours. The reaction was diluted with ethyl acetate (20 mL).
Excess 12 was reduced with 10% Na2S2O7 solution. The organic layer was washed with brine (2 x 10 mL), dried (MgSO4) and concentrated under reduced pressure. Purification by flash column chromatography afforded 60 (0.826 g, 80%) as yellow-orange liquid. IH
NMR (CDCI3): 6.96 (s, IH), 7.18-7.24 (m, 2H), 7.46-7.52) (m, 2H).

3-Iodo-indole (61) [00362] To a mixture of indole (1.0 g, 8.5 mmol) and KOH (1.192 g, 2.5 eq.) in DMF (15 mL) was added 12 (2.38 g, 9.4 mmol) (Scheme 11). After stirring at room temperature for 1 hour, the reaction mixture was poured to an ice-cold solution of 0.1 % Na2SO3 (100 mL), resulting to the precipitation of the product. The product was filtered, dried under high vacuum pressure and used without further purification.
Light orange solid (1.64 g, 80%). 'H NMR (CDC13): 7.19-7.28 (m, 2H), 7.29 (d, 1H, J=2.5), 7.371 (d, 1H, J=7.9), 7.47 (d, 1H, J=7.8).

1-(Toluene-4-sulfonyl) -3-iodo-indole (62) [00363] To a mixture of 3-lodo-indole (0.500 g, 2.05 mmol) and NaOH (0.090 g, 1.1 eq.) in CH2CI2 (10 mL) was added p-toluenesulfonylchloride (0.431 g, 1.1 eq.) (Scheme 11). After stirring at room temperature for 4 hours, the reaction was quenched with a saturated solution of NH4C1(10 mL). Layers were separated. The aqueous layer was extracted with CH2C12 (3 x 8 mL) and the combined organic layer was dried (MgSO4), filtered and concentrated under reduced pressure to give 62 as white solid (0.680 g, 83%). Note that -10% of 3-lodo-indole was recovered. 'H
NMR
(CDC13): 2.35 (s, 3H), 7.21-7.40 (m, 5H), 7.70 (s, 1H), 7.78 (d, 2H, J=8.4), 7.96 (d, 1 H, J=8.7).

Suzuki coupling of 6-hydoxy-2-naphthaleneboronic acid (63) with 59, 60 and 62 [00364] To a degassed solution of 59, 60 or 62 (0.300-0.400 mmol) in DMF (4 mL) was added Pd(OAc)Z (0.05 eq.), K2C03 (2 eq.) and 6-hydroxy-3-naphthaleneboronic acid 63 (1.3 eq.) at room temperature (Scheme 12). After degassing and purging with argon (repeated thrice), the reaction mixture was stirred at 90 C for 2-2.5 hours. The mixture was allowed to cool to room temperature and diluted with H20 (15 mL). The aqueous solution was extracted with ethyl acetate (5 x 15 mL) and combined organic layer was concentrated under reduced pressure. The residue was purified by flash column chromatography using 30 % ethyl acetate in hexane as solvent system to afford 64, 65 or 66.

6-Benzofuran-2 yl-naphthalen-2-ol (64) [003651 Light brown solid (0.049 g, 46%); 'H NMR (CDC13): 7.09 (s, 1H), 7.13-7.17 (m, 2H), 7.21-7.25 (m, 3H), 7.55 (d, IH, J=7.2), 7.59 (d, 1H, J=6.7) 7.74 (d, 1 H, J=7.7), 7.84 (d, 1 H, J=7.6), 7.88 (d, 1 H, J=8.1), 8.31 (s, 1 H).

6-Benzothiophen-2 yl-naphthalen-2-ol (65) [00366] White solid (0.065 g, 61%);'H NMR (DMSO): 7.12-7.20 (m, 2H), 7.36 (t, 1H, J=6.9), 7.40 (t, 1H, J=7.0), 7.77-7.93 (m, 5H), 7.98 (d, 1H, J=7.9), 8.16 (s, 1H);'3C NMR (DMSO): 109.30, 119.94, 119.96, 122.88, 124.04, 124.70, 124.94, 125.25, 127.45, 128.17, 128.34, 130.36, 134.96, 138.89, 141.14, 144.27, 156.58.
6-[]-Toluene-4-sulfonyl)-indol-3 ylJ-naphthalen-2-ol (66) [00367J White solid (0.056 g, 53%);'H NMR (CDC13): 2.33 (s, 3H), 7.14-7.23 (m, 4H), 7.29 (t, 1H, J=7.6), 7.39 (t, IH, J=7.5), 7.63 (dd, 1H, J=1.5, 8.4), 7.72-7.87 (m, 6H), 7.98 (s, IH), 8.07 (s, IH), 8.07 (d, 1H, J=8.3).

6-[indol-3 y1]-naphthalen-2-ol (67) [00368] To a solution of 66 (0.045 g, 0.105mmo1) in MeOH (0.500 mL) was added KOH (0.018 g, 3 eq.) at room temperature (Scheme 12). The reaction mixture was heated at 75 C for 12 hours. The solvent was evaporated under reduced pressure.
Water (7 mL) was added. The aqueous solution was extracted with ethyl acetate (3 x 5 ml). The combined organic layer was washed with a saturated solution of NH4C1 solution (2 x 4 ml), dried (MgSO4), filtered and concentrated under reduced pressure.
The residue was purified by flash column chromatography using 30% ethyl acetate in hexane as solvent system to afford 67 ( 0.20 g, 71.3%) as light yellow solid.
'H NMP.
(acetone-d6): 7.13-7.27 (m, 4H), 7.52 (d, 1H, J=8.0), 7.69-7.88 (m, 4H), 8.06 (d, 1H, J=7.9), 8.14 (s, 1H), 8.55 (s, IH); 13C NMR: 108.92, 111.77, 117.11, 118.48, 119.45, 119.68, 121.69, 122.76, 124.50, 125.86, 126.52, 126.65, 129.13, 129.30, 130.91, 133.43, 137.41, 154.86.

~ ~ B~ OH OH OH
OH ~
HO I / /
63 N 69 HO ~ / 70 Pd(OAc)Z, K2C03 Br DMF, 90 C
-Pd(OAc)20K2C03 ~ ~
66 DMF, 900C N 4M HCI
H
N BOC THF N
,OH

BOC

OH
I
OH
B;OH ~70 Br I ~ ~ OH / ~ HO / / 63 + HO ~ I / Pd(OAc)z, K2C03 74 DMF, 90 C 75 Scheme 13. Syntheses of 6-(Quinolin-3-yl)-naphthalen-2-ol (69), [2,2'1-Binaphthalenyl-6,6'-diol (70), 3-(Indol-2-yl)-quinoline (73) and 6-(Isoquinolin-4-yl)-naphthalen-2-ol (75).

General Procedure for Formation of Aromatic-Aromatic Bond: Suzuki Coupling.

[00369] To a degassed solution of the aryl bromide (68, 74 or 77, Schemes 8 and 9) in DMF (4.0 mL) was added aryl boronic acid (53, 55, 63 or 71, 1.2 equiv), Pd(OAc)2 (0.05 equiv) and K2CO3 (2 equiv) at room temperature. After degassing and purging with argon (repeated thrice), the reaction mixture was stirred at 90 C.
Reaction times vary from 1.5 hours to 12 hours. The mixture was allowed to cool to room temperature and diluted with H20 (15 mL). The aqueous solution was extracted with ethyl acetate (5 x 15 mL) and the combined organic layer was concentrated under reduced pressure. The residue was purified by flash column chromatography.
6-(Quinolin-3 yl)-naphthalen-2-ol (69) [00370] Light yellow solid (0.107 g, 74%). 1H NMR (DMSO): 7.18 (d, 1H, J=8.7 ), 7.21 (s, 1H), 7.67 (t, 1H, J=7.3), 7.78 (t, 1H, J=7.5), 7.86-7.95 (m, 3H), 8.07 (s, 1 H), 8.3 6 (s, 1 H), 8.74 (s, 111), 9.3 9 (s, 1 H), 9.90 (s, 1 H); 13 C
NMR (DMSO):
109.1, 119.8, 125.7, 126.4, 127.5, 127.6, 128.3, 128.5, 128.8, 129.2, 129.8, 130.5, 131.6,132.8,133.4, 134.7,147.1, 150.2,156.5.

[2,2'J-Binaphthalenyl-6,6'-diol (70) [00371] White solid (0.013 g, 9% as side product). 1 H NMR (MeOD): 7.07-7.15 (m, 2H), 7.73 (d, 1H, J=9.16), 7.77-7.82 (m, 2H), 8.06 (s, 1H); 13C NMR
(MeOD): 108.4, 118.3, 124.9, 125.4, 126.4, 128.9, 129.4, 134.1, 136.6, 155.2.
3-(Indol-2-yl)-quinoline (73) [00372] Yellow solid (0.093 g, 56%). 'H NMR (DMSO): 7.06 (t, IH, J=7.4), 7.18 (t, IH, J=7.4), 7.23 (s, 1H), 7.48 (d, IH, J=8.1), 7.62 (d, 1H, J=7.8), 7.67 (t, 1H, J=7.3), 7.77 (t, 1 H, 1=7.1), 8.01 (d, 1 H, J=8.0), 8.05 (d, 114, J=8.0), 8.74 (s, 114), 9.47 (s, 1H), 11.83 (s, 1H); 13C NMR (DMSO): 101.0, 111.9, 120.2, 120.8, 122.8, 126.0, 127.8, 128.1, 128.6, 129.0, 129.3, 129.8, 130.3, 135.2, 138.0, 147.1, 149Ø

6-(Isoquinolin-4 yl)-naphthalen-2-ol (75) [00373] White solid (0.098 g, 75%). 'H NMR (DMSO): 7.18 (dd, 1H, J=2.2, 8.8), 7.24 (d, 1 H, J=2.0, 7.57 (d, 1 H, J=8.4), 7.75 (t, 1 H, J=7.7), 7.80 (t, 1 H, J=7.7), 7.87-7.90 (m, 2H), 7.93 (d, 1H, J= 8.4), 8.53 (s, 1H), 9.37 (s, 1H), 9.9 (s, 1H); 13C
NMR (DMSO): 109.1, 119.7, 124.7, 126.8, 127.9, 128.2, 128.6, 129.1, 130.2, 131.1, 131.5, 133.2, 133.9, 134.6, 143.2, 152.2, 156.4, OH OH
OH

~H KOH ~ } ~
F(O)2 ~ ~
63 ~ \
MeOH/HZ0 ~ ~ COZH
Pd(OAc)2, K2C03 S 78 80 C S 79 DMF, 90 C, 2 h Br B OH
'OH KOH
CJrII>-CO2H
COZR pd(OAc)Z, KZC03 COMe Me O DMF, 90 C, 12 h CH31 76, R= H 80 81 KZC03 ~
77, R = Me /OH
53 'OH
~ O KOH O O
B Q p Pd(OAc)2, KZC03 / ~ ~ S COZMe MeOH/H20 \ COZH
DMF, 90 C, 1.5 h 80 C S

Scheme 14, Syntheses of 3-(6-Hydroxy-naphthalen-2-yl)-benzothiophene-2-carboxylic acid (79), [2,3']-Bi[benzothiophenyl]-2'-carboxylic acid (81) and 3-(Benzofuran-2-yl)-benzothiophene-2-carboxylic acid (83).
3-(6-Hydroxy-naphthalen-2 yl)-benzothiophene-2-carboxylic acid methyl ester (78) [00374] White solid (0.097 g, 78%). 'H NMR (DMSO): 3.71 (s, 314), 7.15 (dd, 1H, J=2.3, J=8.8), 7.23 (d, 1H, J=2.0), 7.39-7.48 (m, 2H), 7.56 (d, 1H, J=8.1), 7.60 (t, 1H, J=7.5), 7.80 (d, 1H, J=8.5), 7.82-7.87 (m, 2H), 8.14 (d, 1H, J=8.1); 13C
NMR

(DMSO): 52.8, 109.1, 119.4, 123.5, 125.5, 125.9, 126.1, 127.7, 127.8, 128.1, 128.5 (2C), 128.9, 130.2, 136.5, 140.0 (2C), 144.2, 156.4, 162.7.
3-(6-Hydroxy-naphthalen-2-yl)-benzothiophene-2-carboxylic acid (79) [00375] White solid (0.085 g, 98%). 'H NMR (DMSO): 7.14 (dd, IH, J=2.3, J=8.8), 7.21 (d, IH, J=2.1), 7.39-7.46 (m, 2H), 7.51 (d, IH, J=8.0), 7.56 (t, 1H, J=7.0), 7.78(d, 1 H, J=8.5), 7.83 (s, I H), 7.84 (d, 1 H, J=8.8), 8.11 (d, IH, J=8.1), 9.83 (s, 111), 13.15 (s, 1H). 13C NMR (DMSO); 109.1, 119.4, 123.4, 125.3, 125.7, 126.0, 127.7, 127.8, 128.7, 128.8, 128.9, 130.0, 130.2, 134.6, 139.9, 140.4, 143.2, 156.3, 163.9;
HRMS: C19H1Z03S calculated 320.0507, found 320.0501.

[2,3 ]-Bi[benzothiophenyl]-2'-carboxylic acid methyl ester (80) [00376] White solid (0.082 g, 72%). 1H NMR (DMSO): 3.83 (s, 3H), 7.35-7.43 (m, 4H), 7.51 (t, 1H, J=7.6), 7.79 (d, 1H, J=7.4), 7.83-7.92 (m, 3H); 13C
NMR
(DMSO): 52.5, 122.2, 122.5, 123.9, 124.5, 125.2, 125.2, 125.4, 127.5, 130.5, 134.8, 135.8, 139.7,140.06,140.1, 140.9,162.5.

[2,3 J-Bi[benzothiophenyl]-2'-carboxylic acid (81) [00377] White solid (0.065 g, 91%). 'H NMR (DMSO): 7.39-7.51 (m, 3H), 7.53-7.62 (m, 2H), 7.7 (d, 1H, J=8.0), 7.94 (d, IH, J=7.2), 8.04 (d, IH, J=7.4), 8.13 (d, 1H, J=8.1), 13.50 (s, IH); 13C NMR (DMSO): 122.7, 123.5, 124.4, 124.9, 125.0, 125.1, 126.0, 126.1, 128.0, 133.0, 134.3, 134.8, 139.5, 139.9, 140.0, 140.5, 163.3;
HRMS: C,7HI002S2 calculated 310.0122, found 310.0122.

3-(Benzofuran-2 yl)-benzothiophene-2-carboxylic acid methyl ester (82) [00378] White solid (0.105 g, 92%). 'H NMR (DMSO): 3.89 (s, 3H), 7.24 (s, 1 H), 7.3 0(t, IH, J=7.4), 7.36 (t, 1 H, J-7.1), 7.5(t, IH, J=7.1), 7.6(d, IH, J=8.2), 7.7 (d, 1H, J=7.7), 7.89 (d, 1H, J=8.1), 8.10 (d, IH, J=8.2); 13C NMR (DMSO): 52.6, 108.8, 111.3, 121.5, 122.5, 123.0, 124.9, 125.4, 125.6, 127.4, 128.5, 130.7, 131.5, 138.8, 140.2, 148.9, 155.0, 162.5.

3- (Benzofuran-2yl)-benzothiophene-2-carboxylic acid ( 83) [00379] White solid (0.094 g, 99%). 'H NMR (DMSO): 7.31- 7.43 (m, 3H), 7.54 (t, 1 H, J=7.9), 7.61 (t, 1 H, J=7.1), 7.68 (d, 1 H, J=8.7), 7.78 (d, 1 H, J=7.3), 7.99 (d, 1H, J=7.5), 8.15 (d, 1H, J=8.4), 13.62 (bs, 1H); 13C NMR (DMSO): 108.7, 111.7, 122.0, 123.59, 123.6, 125.2, 125.4, 126.3, 128.0, 128.7, 130.0, 138.8, 139.6, 148.9, 154.9, 163.3; HRMS: C17HI003S calculated 294.0350, found 294.0350.

Stille Coupling Synthesis of Bis-indoles ~ I I MeOH ~ ~ ~ Iz TsC1, NaH
COOMe D~. ~' i I COOMe H COOH HySOq \ N COOMe DMF
Ts Scheme 15. Preparation of 3-iodo-l-(toluene-4-sulfonyl)-indole-2-carboxylic acid methyl ester (203) Indole-2-carboxylic acid methyl ester (201) [00380] Commercially available indole-2-carboxylic acid 200 (1.61 g, 10 mmol) and concentrated H2SO4 (0.5 mL) were refluxed in dry MeOH (50 mL) for 12 hours. The solution was then cooled to room temperature and concentrated under reduced pressure. Water (25 mL) was added to the residue and adjusted the pH
to 7.

The aqueous layer was extracted with ethyl acetate (3 x 15 mL). The combined organic phase was dried with MgSO4, filtered and concentrated under vacuum to afford 201 (1.75 g 100%). The product was used for the next step without further purification.

3-iodo-indole-2-carboxylic acid methyl ester (202) [00381] To a solution of 201 (0.885 g, 5 mmol) and KOH (0.713 g, 12.5 mmol) in DMF (15 mL) was added I2 (1.396 g, 5.5 mmol) at room temperature. After stirring for 1.5 hours, the reaction mixture was poured to an ice-cold solution of NaZSO3 (0.1%), the precipitation was filtered and dried. The product (1.46 g, 90%) was used for the next step without further purification.
3-iodo-l-(toluene-4-sulfonyl)-indole-2-carboxylic acid methyl ester (203) [00382] To a mixture of 202 (0.604 g, 2 mmol) and NaH (60%, 0.192 g, 2.4 mmol) in DMF (20 mL) was added p-toluenesulfonylchloride (0.381 g, 2 mmol) at room temperature. After stirring for 1 hour, ethyl acetate (50 mL) was added to the reaction mixture. The mixture was washed with brine (3 x 30 mL). The organic layer was dried with MgSO4, filtered and concentrated under vacuum. The residue was purified by flash chromatography (10% ethyl acetate/hexane V:V) to afford 203 (0.68 g, 76%). 'H NMR (CDC13): 2.35 (s, 3H), 4.05 (s, 3H), 7.23 (d, 2H, J=8.4), 7.33 (t, IH, J= 7.2), 7.41, (m. 2H), 7.83 (d, 2H, J=8.4), 7.98 (d, 1H, J=8.4).

COOMe COOMe COOMe 6[N I 12 ~ ~ I II TsC1, NaH \ r 1~
H DMF H DMF j Ts Scheme 16. Preparation of 3-iodo-l-(toluene-4-sulfonyl)-indole-4-carboxylic acid methyl ester (212).

3-iodo-indole-4-carboxylic acid methyl ester (211) [00383] To a solution of commercially available 210 (0.525 g, 3 mmol) and KOH (0.428 g, 7.5 mmol) in DMF (15 mL) was added I2 (0.83 8 g, 3.3 mmol) at room temperature. After stirring for 1.5 hours, the reaction mixture was poured on an ice-cold solution of Na2SO3 (aq., 0.1%). The aqueous layer was extracted with ethyl acetate (3 x 15 mL). The combined organic layers were dried with MgSOa, filtered and concentrated under vacuum. The residue was purified by flash chromatography (3% ethyl acetate/hexane V:V) to afford 211 (0.758 g, 84%). 'H NMR (CDC13):
4.03 (s, 3H), 7.24 (d, 1H, J=7.8), 7.43 (d, 1H, J= 2.5), 7.52 (t, 2H, J=8.5), 8.63 (s, IH).
3-iodo-l-(toluene-4-sulfonyl)-indole-4-carboxylic acid methyl ester (212) [00384J To a mixture of 211 (0.604 g, 2 mmol) and NaH (60%, 0.192 g, 2.4 mmol) in DMF (20 mL) was added p-toluenesulfonylchloride (0.381 g, 2 mmol) at room temperature. After stirring for 1 hour, ethyl acetate (50 mL) was added to the reaction mixture. The mixture was washed with brine (3 x 30 mL). The organic layer was dried with MgSO4, filtered and concentrated under vacuum. The residue was purified by flash chromatography (10% ethyl acetate/hexane V:V) to afford 212 (0.65 g, 71%).

~ I I I_ z~~ I ~ TsCI, NaOli Bu-Li, THF Sneu3 --~ ~
H DMF H CH20Z I Bu3SnCl Ts Ts Scheme 17. Preparation of 3-(tributylstannyl)-I-(toluene-4-sulfonyl)-indole (206) 3-iodo-indole (204) [00386] To a solution of indole (1.0 g, 8.5 mmol) and KOH (1.192 g, 21.25 mmol) in DMF (15 mL) was added 12 (2.38 g, 9.4 mmol) at room temperature.
After stirring for 1 hour, the reaction mixture was poured on an ice-cold solution of Na2SO3 (aq., 0.1%), and the resulting precipitate was filtered and dried. The product (1.83 g, 88%) was used without further purification.

3-iodo-l-(toluene-4-sulfonyl)-indole (205) [00387] To a mixture of 204 (0.5 g, 2.05 mmol) and NaOH (0.09 g, 2.3 mmol) in CHZC12 (10 mL) was addedp-toluenesulfonylchloride (0.431 g, 2.3 mmol) at room temperature. After stirring for 4 h, the reaction was quenched with a saturated solution of NH4C1(aq.) (10 mL). Layers were separated, and the aqueous layer was extracted with CH2CI2 (3 x 8 mL). The combined organic layers were dried with MgSO4, filtered and concentrated under vacuum. The residue was purified by flash chromatography (3% ethyl acetate/hexane V:V) to afford 205 (0.68 g, 83%). 'H
NMR (CDC13): 2.35 (s, 3H), 2.21-7.40 (m, 5H), 7.70 (s, 1H), 7.78 (d, 2H, J=8.4), 7.96 (d, 1H, J=8.7).

3-(tributylstannyl)-1-(toluene-4-sulfonyl)-indole (206) [00388] To a solution of 205 (0.796 g, 2mmol) in THF (20 mL) at -78 C was added butyllithium (1.6 mL, 2.5 M solution, 4 mmol). The solution was stirred for 20 min, and tributyltin chloride (0.56 mL, 2.06 mmol) was added. The mixture was allowed to warm to room temperature and stirred for 4 h. A saturated aqueous solution of KF was poured into the reaction and stirred for 30 min. The aqueous phase was extracted with ethyl acetate (3 x 30mL). The combined organic phase was dried with MgSO4, and the solvent was removed in vacuo to afford 206. The product was used directly for the next step.

sneu3 203, Pd(PPh3)4 LiOH ~
~ ~
~ ~ ~ Cul, DMF i COOMe i ~ THF/MeOH/HZ~ I I I I
O ~ o0H H
Ts Ts Ts Ts LiOH I
THF/MeOH/HZO COOH H

Scheme 18. Preparation of 3-(indole-3-yl)-indole -2-carboxylic acid (209).
3-(1-(toluene-4-sulfonyl)-indol-3 yl)-1-(toluene-4-sulfonyl)-indole-2-carboxylic acid methyl ester (207) [00389] A solution of 203 (0.279 g, 0.613 mmol), crude 206 (0.430 g, 0.766 mmol), a catalytic amount of CuI and tetrakis(triphenylphosphine)palladium in DMF
(20 mL) was degassed with argon for 10 min. The mixture was brought to 90 C
and stirred for 8 h, The solvent was removed in vacuo and the residue was purified by flash chromatography (15% ethyl acetate/hexane V:V) to afford 207. 'H NMR
(CDC13): 2.36 (s, 3H), 2.37 (s, 3H), 3.38, (s, 3H), 7.20 (m, 1H), 7.23-7.27 (m, 4H), 7.32 (d, 2H, J=7.6), 7.35 (d, 2H, J=7.6), 7.43 (m, 1H), 7.78 (s, 1H), 7.83 (d, 2H, J=8.4), 7.89 (d, 2H, J=8.4), 8.04 (d, 1H, J=8.4), 8.11 (d, 1H, J=8.4).

3-(indol-3 yl)-1-(toluene-4-sulfonyl)-indole-2-carboxylic acid (208) [00390] To a solution of 207 (0.2 g, 0.33 mmol) in THF/MeOH/H20 (5:5:1) was added LiOH (0.052 g, 2.2 mmol). The reaction mixture was stirred and refluxed.
After 5 h, the solution was cooled and concentrated. Water (10 mL) was added and pH was adjusted to 2 with 1N HCI. The aqueous phase was extracted with ethyl acetate (3 x l OmL). The combined organic phase was dried with MgSO4, and the solvent was removed in vacuo. The residue was purified by flash chromatography (ethyl acetate/hexane/acetic acid, 30:70:1, V:V) to afford 208 (92 mg, 64%).
'H NMR

(DMSO): 2.34 (s, 3H), 7.07 (t, IH, J=7.5), 7.22 (t, 1H, J=7.5), 7.26 (d, 1H, J=7.7), 7.30-7.32 (m, 2H), 7.36 (t, 1H, J=7.6), 7.42 (d, 2H, J=8.2), 7.53 (d, 1H, J=8.5), 7.89 (s, 1H), 7.93, (d, 2H, J=8.2), 7,99 (d, IH, J=8.3), 11.99 (s, 1H), 12.97 (s, 1H);'3C
NMR (DMSO): 21.5, 111.8, 113.2, 113.7, 116.1, 120.7, 121.2, 121.6, 123.7, 125.0, 125.3, 125.7, 126.2, 127.3, 127.7, 130.7, 131.0, 134.6, 134.7, 136.6, 145.9, 163.1.
3-(indole-3-yl)-indole -2-carboxylic acid (209) [00391] To a solution of 208 (60 mg, 0.14 mmol) in THF/MeOH/H20 (2:5:1) was added LiOH (48 mg, 2 mmol). The reaction mixture was refluxed for 30 h.
The solution was cooled and concentrated. Water (10 mL) was added and pH was adjusted to 2. The aqueous phase was extracted with ethyl acetate (3 x 5mL). The combined organic phase was dried with MgSO4, and the solvent was removed in vacuo. The residue was purified by flash chromatography (ethyl acetate/hexane/acetic acid, 30:70:1, V:V) to afford 209 (30 mg, 78%). 1H NMR (DMSO): 6.97 (t, 1H, J=7.4), 7.02 (t, 1H, J=7.4), 7.11 (t, 1H, J= 7.4), 7.25-7.29 (m, 2H), 7.44-7.50 (m, 4H), 11.27 (s, 1H), 11.68 (s, 1H), 12.68 (s, 1H); 13C NMR (DMSO): 107.5, 111.6, 112.4, 115.5, 118.5, 119.5, 119.9, 120.8, 121.5, 123.9, 124.5, 125.4, 127.0, 127.7, 136.0, 136.2, 163.1.

COOMe COOMe COOH
i ~ ~ SnBu' 212, Pd(PPh3)4 N ~ I I I I\ L---~ ~
1 i 1 ~
~ I CuI, DMF ~ ~ ~ ~ THF/MeOH/HZO ~ H H
Ts Ts Ts Scheme 19. Preparation of 3-(indole-3-yl)-indole -4-carboxylic acid (214) 3-(1-(toluene-4-sulfonyl)-indol-3 yl)-1-(toluene-4-sulfonyl)-indole-4-carboxylic acid methyl ester (213) [00392] A solution of 212 (0.230 g, 0.505 mmol), crude 206 (0.360 g, 0.641 mmol), catalytic amount of Cul and tetrakis(triphenylphosphine)palladium in DMF
(20 mL) was degassed with argon for 10 min. The mixture was brought to 90 C
and stirred for 8 h. The solvent was removed in vacuo and the residue was purified by flash chromatography (15% ethyl acetate/hexane V:V) to afford 213 (210 mg, 70%).
'H NMR (CDC13): 2.36 (s, 3H), 2.38 (s, 3H), 2.43 (s, 3H), 7.14-7.18 (m, 2H), 7.26-7.29 (m, 4H), 7.30-7.34 (m, 1H), 7.41 (t, IH, J=8.0), 7.58 (s, IH), 7.65 (d, 1H, J=7.5), 7.73 (s, 1H), 7.82 (d, 2H, J=8.3), 7.87 (d, 2H, J=8.3), 8.05 (d, IH, J=8.4), 8.25, (d, 1H, J=8.4), 13C NMR (CDC13): 21.6, 21.7, 50.5, 113.6, 114.1, 117.1, 117.3, 120.3, 122.9, 123.6, 124.5, 125.0, 125.6, 125.9, 127.0, 127.1, 127.2, 127.3, 130.0, 130.2, 131.1, 134.8, 134.9, 135.3, 136.0, 145.1, 145.6, 167.7.

3-(indol-3-yl)-indole -4-carboxylic acid (214) [00393] To a solution of 213 (200 mg, 0.334 mmol) in THF/MeOH/HZO
(2:5:1) was added LiOH (48 mg, 2 mmol). The reaction mixture was refluxed for h. The solution was cooled and concentrated. Water (10 mL) was added and pH
was adjusted to 2 with 1N HCI. The aqueous phase was extracted with ethyl acetate (3 x 5mL). The combined organic phase was dried with MgSO4, and the solvent was removed in vacuo. The residue was purified by flash chromatography (ethyl acetate/hexane/acetic acid, 30:70:1, V:V) to afford 214 (50 mg, 54%).'H NMR
(CD3OD): 6.93 (t, IH, J=8.5), 7.06 (t, IH, J=8.5), 7.17-7.21 (m, 2H), 7.35 (d, 1H, J=
8.2), 7.39-7.42 (m, 3H), 7.61 (d, 1H, J=8.2) 10.30 (s, IH); ~3C NMR (CD3OD):
110.3, 110.6, 111.4, 114.4, 118.2, 119.2, 120.0, 120.4, 120.6, 121.9, 124.0, 125.6, 125.7, 128.6, 136.4, 137.7, 172Ø

r>>

In vitro Analysis ofBi-Aromatic Compounds Materials [00394] A(3140 (AnaSpec, San Jose, CA, lots 14212, 34862 and 34889), A(3'"a2 (AnaSpec, lot 45685) and a-synuclein (rPeptide, Bogart, GA, lot 121303AS) were stored at -80 C until used. Positive and negative controls, Thioflavin T, Thioflavin S
and materials for the tris buffers were obtained from Sigma (St. Louis, MO).
Reagents and starting materials for chemical synthesis of bi-aromatics were obtained from Aldrich (St. Louis, MO), Alfa Aesar (Ward Hill, MA) and Combi-Blocks Inc.
(San Diego, CA). All water used in the in vitro studies was micropore filtered and deionized.

[00395] The tau441 expression construct [L Buee et al. (2000). Brain Research Reviews 33:95-130] was purchased (Bioclon Inc., San Diego, CA) already transformed in BL21 D3 bacterial cells (Novagen/EMD Biosciences) for protein expression. Cells were grown and induced with IPTG according to Novagen's protocol. To lyse cells, Cellytic B reagent (Sigma) was used with lysozyme protease inhibitors (Sigma) and benzonase (Novagen/EMD Biosciences), according to manufacturer's methods. Lysate was cleared by centrifugation for 20 min at 10,000 RPM (SS34 rotor, Sorvall RC2b centrifuge) at 4 C. Protein was purified from the soluble fraction. Once purified, tau441 was stored at -78 C as frozen aliquots (8.3 mg/mL, 60 pL) in Tris -HCl (50 mM, pH 7.4) until used.

Stock solutions ofA/11"40, Afl'"41, tau441 and a-synuclein [00396] A[3l-40 (1.0 mg) was pre-treated in a 1.5 mL microfuge tube with 1,1,1,3,3,3-hexafluoroisopropanol (HFIP, I mL) and sonicated for 20 min. to disassemble any pre-formed A[3 aggregates. The HFIP was removed with a stream of argon and the A(3 dissolved in Tris base (5.8 mL, 20 mM, pH 10.5). The pH was adjusted to 7.4 with concentrated HC1 and the solution filtered using a syringe filter (0.2 m). This solution was used for circular dichroism studies or diluted with an equal volume of 8 M Thioflavin T (ThT) in Tris-HCI (20 mM, pH 7.4, 300 mM
NaCI) for the ThT aggregation assay.

[00397] A(3l -42 (1.0 mg) was either pretreated with HFIP in the same manner as A0140 or this step was omitted. After removing HFIP, when appropriate, A[3l-42 was dissolved in 1 /a NH3 (aq.) (200 L) and sonicated for I min. The solution was diluted with Tris-HCI (5.7 mL, 20 mM, pH 7.4), the pH adjusted to 7.4 with concentrated HCI (aq.) and the solution filtered using a syringe filter (0.2 m). Prior to use in the ThT aggregation assay, the solution was diluted with an equal volume of 8 M Thioflavin T (ThT) in Tris-HCI (20 mM, pH 7.4, 300 mM NaCI).

[00398] Frozen aliquots of tau441 (8.3 mg/mL, 60 L) in Tris-HCI (50 mM, pH 7.4) were allowed to thaw at room temperature (RT) before being diluted with Tris-HCI (2.64 mL, 50 mM, pH 7.4) containing dithiothreitol (DTT, 1 mM) to prevent disulfide bonds and NaN3 (50 M) to prevent bacterial growth. After allowing to stand at RT for I h, Thioflavin S (ThS) was added (2.5 L, 10.8 mM), followed by the aggregation inducer heparin (20 L, 1.08 g/mL).

[00399] a-Synuclein (1.0 mg) was dissolved directly in Tris-HCl (11.53 mL, 20 mM, pH 7.4, 100 mM NaC1) containing DTT (5 mM) and ThT (10 M).

(i) Kinetic Thioflavin T/Thioflavin S Assay (LeVine, H., Protein Science, 2:
404-10 (1993)) [00400] Aliquots (200 L) of A(3'40, A[3l "42, tau441 or a-synuclein with ThT
or ThS were added to wells of a black polystyrene 96-well plate, followed by 2 L
of a compound in DMSO (various concentrations), or DMSO alone (controls). For a-synuclein, sodium dodecyl sulfate solution (2.0 L, 30 mM) was also added to each well to induce aggregation. Incubations were performed in triplicate and were taken to contain 20 M A(3'-40 or A(3'-42, 6 M tau441 or 4 M a-synuclein. Plates were covered with clear polystyrene lids and incubated at 37 C in a GENios microplate reader obtained from Tecan Group Limited, Mannedorf, Zurich. For A(3'-40, tau441 and a-synuclein, fluorescence readings (Xe,, = 450 nm, kem = 480 nm) were taken every 15 min., after first shaking at high intensity for 15 sec. and allowing to settle for sec. before each reading. For A(3l -42, readings were taken every 5 min.

[00401] The absorbance of each compound was measured under the conditions used during testing, though on ten times the scale (2 mL), to ensure no significant quenching interfered with the assay. This step was also used to identify whether compounds were completely soluble under testing conditions. Results for those compounds found to absorb significantly (molar absorptivity coefficient at 450 nm greater than 500 cm" M-1) or to not fully dissolve were not used.

"Seeded" Thioflavin T Aggregation Assay for Af3l'40 [00402] The same procedure as described in Thioflavin TAggregation Assay was used for "seeded" ThT aggregarion assay for A(3140 but the HFIP
pretreatment was omitted. The presence of small Ap1-40 aggregates in the untreated batches caused aggregation to begin immediately rather than after a 12-24 hr. lag period.

A(31-40 Disaggregation Assay [00403] A[3r"40 was allowed to aggregate under conditions described in Thioflavin TAggregation Assay except that no DMSO was added initially. After hrs., compound in DMSO or vehicle was added (2 L) and the fluorescence measured every 15 min. as detailed above.

100404] Several bis-indole compounds decreased Ap14O and AR1'12 aggregation, seen as a decrease in ThT fluorescence of at 480nm (Figures 1-5, 7, 8). SDS
was used as a positive control since it is known to stabilize the a-helical, monomeric form of A(31-4 at concentrations above its critical micelle concentration of 8 mM. It was present at 16mM in the SDS incubation. The potent anti-amyloidogenic polyphenol compound morin (Ono, K et al. J. Neurochem, 2003, 87: 172-181) was also used as a positive control.

100405] Bi-aromatic compounds other than bis-indoles were also found to inhibit the aggregation of A~140 and A[i1"42 (Figures 1-5, 7, 8). Aza-indole-containing compounds 10n, On and Oo, for example, as well as compounds containing one or no indoles were found to inhibit aggregation of both isoforms of the peptide (Figures 1C,D,E,F, 7, 8). For indole-phenol compounds, it was found that when indole was linked to two phenol groups (102), it was active at 200 M, whereas when it was linked to a single phenol (103), it was not (Figure 1D). Dose-response activity is shown for some compounds against A(31"4 (Figure 3) and A[314Z (Figure 8) aggregation. All compounds tested against A(3'-40 and AR' -42 were found to have similar activity against both.

[00406] Figure 1 depicts the inhibition of A[31-40 aggregation, as measured by Thioflavin T (ThT) fluorescence, by directly-linked 3,3'-bis-indolyl compounds Oc-i (A), other bis-indoles (B), aza-indole-containing compounds (C), indole-phenols (D), indole-5-carboxylic acid-containing bi-aromatics (E), and naphthol-containing bi-aromatics (F). Aggregation conditions: 20 M A(3l-40 incubated in covered black 96-well polystyrene microplates with 4 M ThT, pH 7.4, Tris-HC1(20 mM), 150 mM

NaCI, 1% DMSO. Compound concentrations are 200 M (A,B, except where otherwise noted), 100 M (E), 20 M (F), or as indicated (C,D). Plates heated at 37 C in Tecan Genios microplate reader. All incubations performed in triplicate.
Prior to experiment, A(3140 pretreated with 1, 1, 1,3,3,3-hexafluoroisopropanol (HFIP).
Fluorescence readings: fluorescence measured every 15 min. (%e7C = 450 nm, %em =
480 nm) after shaking for 15 sec. and pausing 10 sec. before taking measurement.
Values are the mean of three replicates. Error bars, omitted for clarity in (B), represent standard deviation of the mean.

[00407] Figure 2 depicts dose-response effect of Oc on A[3'-40 aggegation in kinetic ThT assay. Figure 3 depicts dose-response curves for inhibition of A[3l -40 aggregation by Oj and positive control, morin. Figure 4 depicts the inhibition of Apl"
4o aggregation by Oc in "seeded" ThT assay. Figure 5 depicts disaggregation of A(3l-40 by Oc and positive control, morin. After incubating at 37 C for 46 hrs. Oc, morin or vehicle were added as DMSO solutions (1:100) and the fluorescence measured every 15 min.

[00408] Figure 7 depicts inhibition of A[3142 aggregation by various synthesized bi-aromatic compounds and positive controls morin (Ono K, Yoshiike Y, Takashima A, Hasegawa K, Naiki H, Yamada M. 2003. J Neurochem 87:172-8 1) and RS-0406 (Walsh DM, Townsend M, Podlisny MB, Shankar GM, Fadeeva JV, Agnaf OE, Hartley DM, Selkoe DJ. 2005. J Neurosci 25:2455-62) in the kinetic ThT
assay.
Same conditions as for A(31"40, Figure 1. Compound concentrations were 100 M
in (A), 20 M in (B), except for RS-0406, which was tested at 100 M, and 20 M
in (C).

[00409] Figure 8 depicts dose-response curves for compounds Oj, Ok and 64 at inhibiting aggregation of Apl '42 in the kinetic ThT assay.

(ii) Circular dichroism studies [00410] Aliquots (220 L) of pretreated A(3140 (40 M in 20mM Tris-HCI, pH
7.4) were added directly to 1 mm quartz circular dichroism (CD) cells, followed by 2.2 L compound (of varying concentration) in methanol or methanol alone (controls).
Solutions were incubated at 37 C for up to 6 days. CD scans were performed on a J-810 spectropolarimeter (obtained from Jasco Corporation, Tokyo, Japan) between and 250nm, with a resolution of 0.1nm and bandwidth of Inm. Ten scans were obtained for each reading. Spectra had background CD of the buffer subtracted and were left unsmoothed. Readings were performed at 37 C.

[004111 A(3l'40, in the absence of any compound, shifted from primarily random coil (RC) at the beginning of an experiment to primarily (3-sheet over the course of about 3 days (Figure 6A). When a bis-indole compound was added (e.g. Oc, Oj), this transition was inhibited (Figure 6B). In the case of Oj, the RC->(3-sheet transition was inhibited to a greater extent than by the potent anti-amyloidogenic polyphenol morin (Ono K, Yoshiike Y, Takashima A, Hasegawa K, Naiki H, Yamada M. 2003. J
Neurochem 87:172-81) (Figure 6B, C).

[00412] Figure 6 depicts circular dichroism (CD) of A[i140 alone (A) and in the presence of Oj (B) and morin (C). Conditions: 40 M A[31-40, 200 M compound (B,C), pH 7.4, Tris-HCl (20 mM), 1% MeOH. Prior to experiment, A[it-40 pretreated with 1, 1, 1,3,3,3-hexafluoroisopropanol (HFIP).

(iii) IH NMR Binding of Bi-aromatics to Ap I'40 [00413] AR'40 was dissolved in HFIP (1 mL) and sonicated (20 min) to disassemble any pre-formed aggregates. The HFIP was removed with a stream of Ar(g) and the waxy residue dissolved in 2.2 mL buffer (Tris-dil, 20 mM in D20).
Incubations (0.500 mL) were made directly in thin-walled NMR tubes, to which were added compound (2.5 L, 10 mM in DMSO-d6) or vehicle. An identical incubation lacking A[i1-40 was used as compound reference. Spectra were obtained for A43'-alone (100 M), compound alone (50 M) and for a mixture of both (100 M
A(3140, 50 M compound). Spectra were recorded at 500 MHz and 27 C (300K), with 674 scans obtained for each.

[00414] A subset of active compounds were evaluated in the 'H NMR binding experiments (Figure 9). The proton peaks of compounds experienced pronounced signal broadening in the presence of Ap140 (2:1 peptide:compound), a phenomenon characteristic of ligand-protein binding (Zartler ER, Yan J, Mo H, Kline AD, Shapiro MJ. 2003. Curr Topics Med Chem 3:25-37).

[00415] Figure 9 depicts 'H NMR spectra identifying binding of 5-bromo-5'-carboxy-3,3'-bis-indolyl (0c) to A(3140. Spectra were obtained for A(31-40 alone (100 M, A), compound alone (50 M, C) and for a mixture of both (100 M A(3'-40, 50 M compound, B). Spectra were recorded at 500 MHz and 27 C (300K) in D20 containing 1% DMSO-d6, and 674 scans were obtained for each.

(iv) tau441 Aggregation [00416] A kinetic ThS assay of tau441 aggregation was used to evaluate the bi-aromatic compounds, as detailed in Example 8, paragraph (i). Some synthesized compounds both reduced the rate of tau fibrillization and the equilibrium or plateau level of fibrillization (Figure 10, A,B,C,E). This was also found to be the case for the positive control morin (Figure 10D); polyphenols such as morin have been shown to inhibit tau fibrillization (Taniguchi S, Suzuki N, Masami M, Hisanga S, Iwatsubo T, Goedert M, Hasegawa M. 2005. J Biol Chem 280:7614-7623). All compounds in Figure 10 inhibit A(3 aggregation in a Thioflavin T fluorescence assay and therefore have potential for a dual mechanism of action for ameliorating Alzheimer's disease.
[00417] Figure 10 shows the inhibition of tau fibrillization by synthesized bi-aromatic compounds and morin. Aggregation conditions: tau441 at a concentration of either 10 M (A) or 4 M (B,C,D,E) was incubated in covered black 96-well polystyrene microplates alone or with 100 (A) or 50 M compound (B,C,D,E), 5 M
ThS, pH 7.4, Tris-HCI (50 mM) containing NaN3 (50 M), and either 1% MeOH (A) or 1% DMSO (B,C,D,E). Plates were heated at 37 C in a Tecan GENios microplate reader. All incubations performed in triplicate, except for compound Oc in (A), which had n=l. Fluorescence readings: fluorescence measured every 15 min. (AeX = 450 nm, kem = 480 nm) after shaking for 15 sec. and pausing 10 sec. before taking measurement. Values are the mean of three replicates. Error bars, sometimes not visible due to small size, represent standard deviation of the mean.

[00418] Unlike those compounds of Figure 10, some compounds were found to increase the initial rate of tau aggregation, though had lower plateaus than the control (Figure 11). The lower equilibrium level of aggregation may indicate a decreased amount of tau fibrillization and suggests these compounds may still have benefit in treating AD. As with Figure 10, all compounds in Figure 11 were found to inhibit A(3 aggregation. This demonstrates variable activity of the compounds against different misfolding proteins. In other words, a given bi-aromatic compound can inhibit two or more proteins/peptides from aggregating, or it may inhibit the aggregation of one while promoting that of another.

[00419] Other than 83 in Figure 10E, all the bi-aromatics tested significantly modulated tau fibrillization at concentrations of 50 to 100 gM (Figures 10 and 11).

Conversely, nicotinic acid, tested as a negative control, had no effect on tau fibrillization, even at a concentration of 1 mM (Figure 11D). This demonstrates the specificity of binding of bi-aromatic compounds to tau.

[00420] Figure 11 shows the effect on tau aggregation of synthesized bi-aromatic compounds and nicotinic acid. Aggregation conditions: tau441 at a concentration of 4 M was incubated in covered black 96-well polystyrene microplates alone or with 50 M (A,B,C) or 1 mM compound (D), 5 M ThS, pH
7.4, Tris-HCI (50 mM) containing NaN3 (50 M), and 1% DMSO. Plates were heated at 37 C in a Tecan GENios microplate reader. All incubations performed in triplicate.
Fluorescence readings: fluorescence measured every 15 min. (1,ex = 450 nm, 2,,,m =
480 nm) after shaking for 15 sec. and pausing 10 sec. before taking measurement.
Values are the mean of three replicates. Error bars, sometimes not visible due to small size, represent standard deviation of the mean.

[00421] Heparin is used as an inducer of aggregation in the ThS tau assay (Figures 10, 11). When it was omitted, tau aggregation was still found to occur, but at a much slower rate. Compound Oc and positive control morin both inhibited tau aggregation in the absence of heparin inducer.

(v) a-Synuclein Aggregation [00422] Aggregation of the a-synuclein protein has been implicated in the pathogenesis of a number of neurodegenerative conditions, including Alzheimer's disease, Lewy body diseases (e.g. Parkinson's disease) and multiple system atrophy (Ono K, Yamada M. 2006. J Neurochem 97:105-15). Inhibiting its aggregation is expected to be beneficial in treating these diseases. Compounds were evaluated for their ability to inhibit a-synuclein aggregation using a ThT fluorescence assay similar to that of Necula M, Chirita CN, Kuret J. 2003. J Biol Chem 21:46674-80.
Several of the synthesized bi-aromatic compounds, as well as positive controls, were found to be active in the assay (Figures 12).

[00423] Figure 12 depicts inhibition of a-synuclein aggregation by synthesized bi-aromatics and positive control morin (K, Yamada M. 2006. J Neurochem 97:105-15). Aggregation conditions: a-Synuclein (6 M) was incubated in covered black well polystyrene microplates in Tris-HC1(20 mM, pH 7.4), 10 M ThT, 1% DMSO
with compound concentrations of 100 M (A) or as indicated (B). Plates were heated at 37 C in a Tecan Genios microplate reader. All incubations were performed in triplicate. Fluorescence readings: fluorescence was measured every 15 min. (~, =
450 nm, ~em = 480 nm) after shaking for 15 sec. and pausing 10 sec. before taking measurement. Values are the mean of three replicates. Error bars represent standard deviation of the mean.

Trp-Trp Dipeptides as Therapeutic Agents [00424] The Trp-Trp dipeptides synthesized (Table 2) have two benefits as therapeutic agents: 1) They are unlikely to possess significant toxicity, given that they are composed of a naturally occurring amino acid and/or its enantiomer i.e. L-or D-tryptophan. 2) Due to their similarity to L-Trp they may be recognized by the large neutral amino acid transporter and thereby cross the blood-brain barrier.
These two characteristics increase the likelihood of the dipeptides having favourable pharmacokinetics.

[00425] Certain Trp-Trp dipeptides in accordance with the present invention are listed in Table 2 below.

(a) Linear Trp-Trp dinentides (b) LXclic Tr-TdipeUtides W X H X' ~N COZ W NH
+ O Y K11Z HN ~IIY

Z
O
W X Y Z
H H
L-L ~~ " H H i~ N W X Y Z
L D i~ r"r H " H cyclo(L-L) i. N H H i. N
) ~
~j i ) -11 .
H H
H
D-L H H cyclo(D-D) H N 1%
H ID N H iD N

H
D-D H H meso-cyclo ~. N H ~~ N H
H I~ " Q,- N. H ~

[004261 Since any anticipated drug-receptor binding is dependent on the three-dimensional structure of the drug's interacting functional groups, all combinations of L- and D-stereoisomers of Trp-Trp were synthesized (L-L, L-D, D-L, D-D). This served the dual purposes of increasing the likelihood of producing a successful interaction and providing possible clues as to the stereochemical requirements of binding. Incorporating the D-enantiomer of Trp into the molecule was also part of the overall peptidomimetic drug-design process. Since the D-enantiomer is non-naturally occurring, the chance of enzymatic amide bond cleavage of D-containing peptides is reduced, thereby resulting in improved pharmacokinetics over the L-L dipetide.
[00427] The synthesis of cyclic Trp-Trp dipeptides was also performed as a further step in the peptidomimetic process. Possessing neither an amino nor carboxyl terminus, these molecules have a reduced likelihood of being metabolized by endogenous proteases. Another attractive feature of cyclic dipeptides is their general lack of toxicity. Their cyclic structure limits conformational freedom and therefore reduces the number of non-target receptors with which they can interact.
Finally, many cyclic peptides are capable of crossing the blood-brain barrier due to their lipophilicity and lack of zwitterionic termini, making them suitable for the treatment of neurological diseases.

Synthesis of Trp-Trp Dipeptides [00428] The synthesis of the tryptophan dipeptides first involved coupling of Trp, protected at the N-terminal with the benzyloxycarbonyl (CBZ) group, to Trp protected at the C-terminal by a methyl ester. Coupling of one of the N-protected residues (either D or L) to one of the C-protected residues was carried out using the coupling reagent PyBOP and HOBt. (PyBOP = Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; HOBt = N-Hydroxybenzotriazole). This step is shown in Scheme 20 of Example 10 for the formation of protected L-L. The protecting groups were then removed via the saponification of the methyl ester and catalytic hydrogenation of the CBZ
group to give the linear dipeptides.

[00429] To produce cyclic dipeptides, catalytic hydrogenation was performed on diprotected Trp-Trp to remove the C$Z group, followed by a reflux in methanol to effect the intramolecular cyclization. The reflux was performed in the presence of the base 1,4-diazabicyclo[2.2.2]octane (DABCO) to enhance the nucleophilicity of the N-terminal nitrogen and thereby speed the reaction rate. Ammonia has been used in this capacity in earlier syntheses of cyclo(L-Trp-L-Trp). The saponification, hydrogenation, and cyclization reactions are presented in Scheme 21 of Example 10.

H
N \

H
~\H CN
O~H COOH

(CBZ-Trp) PyBOP, HOBt 0 0 H N
+ \/ C(Oh1'N ~ON

\ OC') N
H
H
+ OMe (CBZ-Trp-Trp-OMe) Cl- H3N

(Trp-OMe = HCI) Scheme 20. General coupling procedure in the synthesis of Trp-Trp. The reaction shown above is for the L-L isomer of the dipeptide.

[00430] While a total of four stereoisomers exist for linear Trp-Trp (L-L, L-D, D-L and D-D), only three different configurations exist for cyclic Trp-Trp, namely cyclo(L-L), cyclo(D-D) and meso-cyclo(Trp-Trp); upon cyclization, the carboxyl and amino terminal residues become indistinguishable from one another and as a result, the L-D and D-L isomers are rendered identical. The three isomers of cyclic Trp-Trp are shown in Table 2 above and their general synthesis from the corresponding diprotected dipeptides is given in Scheme 21 of Example 10.

H
N
O
N~ H H OII
O N '.OMe N~
H O H NaOH, MeOH I O H OH

O
\
H / N
H
H2, Pd/charcoal MeOH
H2, Pd/charcoal MeOH
H
N \
H
I / N \
O
HzN N~OMe H H O
HZN N~OH
~ H H
&~N
&~N
A MeOH, DABCO L-L
H O H
N N
I 13i~.. NH I
O

cyclo(L-L) Scheme 21. General synthesis of cyclic and linear Trp-Trp from diprotected dipeptides. The reaction shown above is for the L-L isomer. DABCO: 1,4-diazabicyclo[2.2.2]octane.

Experimental [00431] Reagents and solvents were obtained from commercial sources (Aldrich, Bachem, and Fluka). Melting points (mp) were determined using a Mel-Temp II capillary apparatus and are uncorrected. Optical rotation was measured at the sodium D-line (589nm), using an Autopol II automatic polarimeter with a path length of 1 decimeter; values are reported in units of degrees mL g"1 dm'1. Thin layer chromatography (TLC) was performed using coated Brinkmann silica gel 60 F254 plates with aluminum backing. Solvent systems used for TLC are given in Table 3.
Compounds were visualized using UV light. The presence of primary amine functional groups was determined by developing the plates with ninhydrin.

[00432] Infrared (IR) spectra were recorded on a Bomem 120 FT-IR
spectrophotometer using KBr disks. 1H and 13C NMR spectra were recorded with a Bruker Avance 400 MHz spectrometer in deuterated dimethylsulfoxide (DMSO-d6).
Chemical shifts (S) are reported as parts per million downfield of tetramethylsilane (TMS) and are calibrated based on solvent peaks. Correlation spectroscopy techniques (COSY and HETCOR) were used to confirm structural connectivity.
[00433] Fast atom bombardment (FAB) mass spectrometry was performed on a VG Quattro mass spectrometer. Compounds were analyzed in 2% acetic acid in glycerol. High performance liquid chromatography (HPLC) was performed on a System Gold apparatus from Beckman fitted with a C 18 reverse phase column.
Methanol and 0.2% trifuoroacetic acid (TFA), both HPLC grade, were used for the solvent system and compound detection was achieved by monitoring absorbance at 220nm. All compounds synthesized were greater than 95% pure using the HPLC
method outlined above.

Solvent systems used for TLC.

Solvent system Solvents Ratio A Acetonitrile / water / acetic acid 20:1:1 B Ethanol / acetic acid 50:1 C Acetonitrile / water / acetic acid 4:1:1 D Ethyl acetate / methanol 5:1 General procedure for coupling of CBZ-Trp and Trp-OMe.
[00434] The N-protected Trp (0.679g, 2.01 mmol, 1.0 eq.) was dissolved in THF (30 mL). PyBOP (1.0 eq.) was added followed by HOBt (1.0 eq.) and the carboxylic acid protected amino acid salt (0.563g, 2.20 mmol, 1.1 eq.).
Diisopropylethylamine (2.1 eq.) was added and the mixture stirred for 24 hrs (TLC
completion). The solution was concentrated under reduced pressure and the resulting oil was dissolved in ethyl acetate (EtOAc, 25 mL). The organic phase was washed with brine (3x20 mL), 1M HCI (2x20 mL), saturated NaHCO3 (3x20 mL) and finally with brine again (3x20 mL). The organic phase was dried over MgSO4, filtered and concentrated. The resulting yellow oil was recrystallized from EtOAc/hexanes and dried in vacuo, yielding a white solid. The yields, melting points and optical rotations for the compounds are shown in Table 4, along with literature values (all four diasteromers have been previously synthesized and characterized in Guarnaccia, et al., Biopolymers 1975, 14, 2329-2346; Chieu, et al., Journal of the American Chemical Society 1982, 104, 3002-3007). Other physical properties of the diprotected dipeptides (e.g. spectra from 'H and 13C NMR, IR and mass spectrometry) were as expected and conformed to literature findings.

H
N
\ I ~
O O
H
O~H OMe &~N
H
CBZ-Trp-Trp-OMe Physical properties of CBZ-Trp-Trp-OMe isomers.
Compound Yield Melting point ( C) [a]pZ5 Found Lit. Found Lit.
CBZ-L-Trp-L-Trp-OMe 86% 192-194 194-197 +23.5 +27.1 CBZ-L-Trp-D-Trp-OMe 87% 197-201 199-201 -13.9 -CBZ-D-Trp-L-Trp-OMe 92% 211-214 205-206 +14.2 -CBZ-D-Trp-D-Trp-OMe 89% 192-194 196-198 -21.2 -All values of [a]DZ5 measured in glacial acetic acid (AcOH), at a concentration of 0.8 g/dl (See Chieu DT et al. 1982. J Am Chem Soc 104:3002-7) (See also Guaraccia R et al. 1975. Biopolymers 14:2329-46) General procedure for removal of methyl ester from the C-terminal of Trp-Trp.
[00435] The saponification method of McDermott et al., Journal of the American Chemical Socfety 1982, 104, 3002-3007 was used. The diprotected dipeptide (0.425g, 0.79 mmol) was dissolved in MeOH (20mL) and I N NaOH (5 mL) was added to the solution. On occasion, the reaction was performed on double or triple this scale without difficulty. The reaction was stirred and monitored by TLC.
After 2 hrs., water (20 mL) was added and unreacted compound was removed by washing with ether (2x20 mL). The aqueous layer was then acidified to pH 2 with cone. HC1 and EtOAc was quickly added (20 mL). The EtOAc layer was separated and the aqueous layer extracted with two more portions of EtOAc (20 mL). The extracts were dried over MgSO4, concentrated and the resulting oil was recrystallized from EtOAc/hexanes and then from MeOH/H2O. The white solid obtained was dried in vacuo.

O(H
~
D
O C H O
p H B N OH
A O F
/ 1 \
NH
CBZ-Trp-Trp-OH

[00436] The four diastereomers of the N-protected dipeptides (CBZ-Trp-Trp-OH) in accordance with the present invention were as follows:
CBZ-L-Trp-L-Trp-OH

[00437] White solid (0.257g, 87.3%); mp 207-210 C (lit. 209-210 C); [aJpzs +19.2, c 0.8, AcOH (lit. +23.7, c 0.8, AcOH); TLC (Rf) A: 0.74, B: 0.69; IR
(vmZ"):
3412 (NH), 3372 (NH), 3334 (NH), 3054 (CH, aromatic), 2921 (CH), 1729 (COOH), 1695 (C=O), 1659 (C=O), 1515 (C=C, aromatic) cm"';'HNMR: 8: 2.90 (dd, 1H, Hc, J=11.7 Hz, J=12.1 Hz), 3.21 (m, 3H, 2HF and Hc,), 4.35 (m, 1H, HB), 4.54 (m, IH, HE), 4.94 (s, 2H, Z-CH2), 7.2 (m, 14H, 13Ar-H and HA), 7.56 (d, 1H, Ar-H, J=7.4 Hz), 7.65 (d, 1H, Ar-H, J=7.5 Hz), 8.29 (d, 1H, HD, J=7.1 Hz), 10.82 (s, 1H, indole N-H), 10.90 (s, 1H, indole N-H), 12.64 (bs, 1H, CO2-H);13C NMR: 28.02, 28.69, 53.82, 56.19, 66.09, 110.49, 111.05, 112.16 (2C), 116.70 (2C), 119.04, 119.25, 119.42 (2C), 121.68, 121.78, 124.69 (2C), 128.12, 128.31, 128.51, 129.15, 136.91, 137.81, 153. 21 (2C), 156.64, 172.81, 174.13; FAB (m/z): 525.2 [M+H]+.

CBZ-L-Trp-D-Trp-OH
[00438] White solid (0.799g, 81.6%); mp 184-186 C; [a]DZ5 -11.7, c 0.8, AcOH; TLC (Rf) A: 0.74, B: 0.69; IR (vR,.): 3409 (NH), 3334 (NH), 3058 (CH, aromatic), 2934 (CH), 1715 (COOH), 1667 (C=0), 1515 (C=C, aromatic) cm"1: 'H

NMR S: 2.72 (dd, 1H, Hc, J=14.4 Hz, J=10.3 Hz), 2.90 (dd, 1H, Hc,, J=10.6 Hz, J=3.1 Hz), 3.03 (dd, 1H, HF, J=8.5 Hz, J=14.5 Hz), 3.17 (dd, 1H, HT, J=4.9 Hz, J=14.4 Hz), 4.33 (m, 1H, HB), 4.50 (m, 1H, HE), 4.92 (s, 2H, Z-CH2), 7.2 (m, 14H, 13Ar-H
and HA), 7.55 (d, 1H, Ar-H, J=7.8 Hz), 7.60 (d, 1H, Ar-H, J=7.9 Hz), 8.32 (d, 1H, HD, J=7.9 Hz), 10.74 (s, 1H, indole N-H), 10.85 (s, 1H, indole N-H), 12.71 (bs, 1H, C02-H); 13C NMR: 28.02, 28.69, 53.69, 56.08, 65.97, 110.49, 110.95, 111.97, 112.10, 118.92, 118.99, 119.16, 119.44, 121.54, 121.68, 124.50, 124.61, 127.95, 128.03, 128.19, 128.40, 129.05 (2C), 136.79, 136.85, 137.75 (2C), 156.51, 172.54, 174.07;
FAB (m/z): 525.2 [M+H]+.

CBZ-D-Trp-L-Trp-OH
1004391 White solid (0.829g, 83.5%); mp 189-191 C; [a]DZ5 +12.4, c 0.8, AcOH; TLC (Rf) A: 0.74, B: 0.69; IR (vma),): 3407 (NH), 3334 (NIH), 3057 (CH, aromatic), 2926 (CH), 1714 (COOH), 1666 (C=0), 1517 (C=C, aromatic) cm"1 : 1H
NMR S: 2.72 (dd, 1H, Hc, J=14.4 Hz, J=10.4 Hz), 2.90 (dd, 1H, Hc,, J=3.7 Hz, J=14.4 Hz ), 3.04 (dd, 1 H, J=8.5 Hz, J=14.5 Hz), 3.17 (dd, 1 H, J=4.9 Hz, J=14.4 Hz), 4.33 (m, 1H, H$), 4.51 (m, 1H, HE), 4.92 (s, 2H, Z-CH2), 7.2 (m, 14H, 13Ar-H and HA), 7.5 5(d, 1 H, Ar-H, J=7.8 Hz), 7.60 (d, 1 H, Ar-H, J=7.9 Hz), 8.32 (d, IH, HD, J=7.9 Hz), 10.74 (s, IH, indole N-H), 10.85 (s, 1H, indole N-H), 12.71 (bs, 1H, C02-H);13C
NMR: 28.03, 28.70, 53.69, 56.09, 65.97, 110.49, 110.95, 111.97, 112.11, 118.92, 118.99, 119.16, 119.44, 121.54, 121.68, 124.50, 124.61, 127.95, 128.03, 128.19, 128.40, 128.58, 129.05, 136.79, 136.86, 137.75 (2C), 156.51, 172.55, 174.08;
FAB
(m/z): 525.2 [M+H]+.

CBZ-D-Trp-D-Trp-OH

[00440] White solid (0.838g, 86.2%); mp 206-207 C; [a]DZS -17.5, c 0.8, AcOH; TLC (Rf) A: 0.74, B: 0.69; IR (vma,,): 3409 (NH), 3334 (NH), 3057 (CH, aromatic), 2934 (CH), 1716 (COOH), 1663 (C=0), 1519 (C=C, aromatic) cm"1; 'H
NMR: 8: 2.90 (dd, IH, Hc, J=4.0 Hz, J=14.6 Hz), 3.21 (m, 3H, 2HF and Hc,), 4.35 (m, 1H, HB), 4.54 (m, 1H, HE), 4.94 (s, 2H, Z-CH2), 7.2 (m, 14H, 13Ar-H and HA), 7.56 (d, I H, Ar-H, J=7.4 Hz), 7.65 (d, 1 H, Ar-H, J=7.8 Hz), 8.29 (d, 1 H, HD, J=7.4 Hz), 10.82 (s, 1H, indole N-H), 10.90 (s, 1H, indole N-H), 12.73 (bs, 1H, C02-H);

NMR: 27.87, 28.63, 53.82, 56.21, 66.09, 110.49, 111.04, 112.21 (2C), 116.67 (2C), 119.04, 119.25, 119.43 (2C), 121.69, 121.78, 124.52, 124.70, 128.12, 128.31, 128.51, 129.16, 136.91, 137.81, 152.69, 155.01, 156.64, 172.82, 174.14; FAB (m/z):
525.2 [M+H]+.

General procedure for removal of CBZ group from the N-Terminal of Trp-Trp.
[00441] The CBZ group was removed using catalytic hydrogenation. The N-protected dipeptide (0.470g, 0.89 mmol) was dissolved in MeOH (20 mL) and the system flushed with N2. Next, 10% palladium on charcoal (0.470g) was added and the system was flushed with N2 followed by H2. The mixture was then stirred vigorously under H2 pressure from a balloon until TLC indicated completion (approximately 1 hour). The solution was filtered and concentrated. The resulting clear oil was recrystallized from MeOH /.Et20 /hexanes, and dried in vacuo, giving a white or light pink powder.

H
N

D
C H O
HZN g N OH
A O F

H
H-Trp-Trp-OH

[004421 The four diastereomers of H-Trp-Trp-OH in accordance with the present invention were as follows:

H-L-Trp-L-Trp-OH
[004431 White solid (0.285g, 81.1%); mp 174-176 C (lit.186 C); [a]p25 -9.0, c 0.8, EtOH (lit. -11.0, c 0.426, EtOH);TLC (Rf) B: 0.30, C: 0.71; IR (vmax):

(NH), 3059 (CH, aromatic), 2927 (CH), 1668 (C=0), 1600 (C=O), 1522 (C=C, aromatic) cm"': 'H NMR S: 2.84 (dd, 1H, Hc, J=8.5 Hz, J=14.9 Hz), 3.12 (m, 3H, and Hc,), 3.62 (dd, IH, HB, J=4.4 Hz, J=8.2 Hz), 4.51 (m, 1H, HE), 5.9 (bs, 2H, HA), 7.2 (m, 10H, Ar-H), 8.3 5 (d, 1 H, HD, J=7.4 Hz), 10.84 (s, 1 H, indole N-H), 10.97 (s, 1H, indole N-H);13C NMR: 28.25, 30.37, 54.15, 55.12, 110.24, 110.74, 111.98, 112.16, 118.92, 119.06, 119.27 (2C), 121.49, 121.71, 124.37, 125.01, 128.19, 128.38, 136.77, 137.09, 173.02, 174.16; FAB (m/z): 391.3 [M+H]+.

H-L-Trp-D-Trp-OH
[004441 Light pink solid (0.261g, 75.0%); mp 173-176 C; [a]DZS +32.0, c 0.8, EtOH; TLC (Rf) B: 0.17, C: 0.62; IR (vmax): 3407 (NH), 3059 (CH, aromatic), (CH), 2855 (CH), 1668 (C=0), 1600 (C=O), 1526 (C=C, aromatic) cm"1;'H NMR 8:

2.69 (dd, IH, Hc, J=9.1 Hz, J=14.4 Hz), 3.04 (m, 2H, Hc, and HF), 3.19 (dd, 1H, HF, J=5.5 Hz, J=15.1 Hz), 3.73 (m, 1H, HB), 4.45 (m, 1H, HE), 6.11 (bs, 2H, HA), 7.3 (m, lOH, Ar-H), 8.37 (s, 1H, HD), 10.82 (s, 1H, indole N-H), 10.92 (s, 1H, indole N-H);
13C NMR: 28.47, 29.98, 54.74 (2C), 109.90, 111.21, 111.98, 112.11, 118.89, 119.01, 119.23, 119.29, 121.44, 121.67, 124.35, 125.07, 127.99, 128.41, 136.80, 137.04, 171.98, 174.60; FAB (m/z): 391.3 [M+H]+.

H-D-Trp-L-Trp-QH
[00445] Light pink solid (0.310g, 85.0%): mp 179-182 C: [a]D25 -40.3, c 0.8, EtOH; TLC (Rf) B: 0.17, C: 0.62; IR (v ,.): 3407 (NH), 3262 (NH), 3059 (CH, aromatic), 2921 (CH), 1671 (C=0), 1600 (C=O), 1526 (C=C, aromatic) cm 1: 'H
NMR S: 2.68 (dd, 1H, Hc, J=9.0 Hz, J=14.2 Hz), 3.02 (m, 2H, Hc, and HF), 3.18 (dd, IH, HF, J=4.4 Hz, J=14.1 Hz), 3.71 (m, 1H, HB), 4.44 (m, 1H, HE), 5.50 (bs, 2H, HA), 7.3 (m, l OH, Ar-H), 8.37 (s, 1 H, HD), 10.81 (s, 1 H, indole N-H), 10.90 (s, 1 H, indole N-H);13C NMR: 28.41, 30.09, 54.64, 54.76, 109.94, 111.10, 111.99, 112.10, 118.91, 119.01,119.20,119.28,121.46,121.67,124.36,125.04,127.99,128.36,136.80, 137.03, 172.14, 174.47; FAB (m/z): 391.3 [M+H]+.

H-D-Trp-D-Trp-OH
[00446] White solid (0.351g, 73.1%); mp 172-175 C: [a]D25 +11.0, c 0.8, EtOH; TLC (Rf) B: 0.30, C: 0.71; IR (vm~: 3406 (NH), 3059 (CH, aromatic), 2927 (CH), 2868 (CH), 1668 (C=0), 1600 (C=0), 1523 (C=C, aromatic) cm"t:'H NMR S:
2.82 (dd, 1H, Hc, J=8.5 Hz, J=14.2 Hz), 3.13 (m, 3H, 2HF and HC.), 3.62 (m, 1H, HB), 4.52 (m, 1H, HE), 4.70 (bs, 2H, HA), 7.2 (m, IOH, Ar-H), 8.39 (d, 1H, HD, J=6.8 Hz), 10.83 (s, IH, indole N-H), 10.94(s, 1H, indole N-H); 13C NMR: 28.27, 30.38, 54.15, 55.12, 110.26, 110.75, 111.98, 112.16, 118.93, 119.08, 119.27 (2), 121.50, 121.71, 124.37, 125.04, 128.20, 128.38, 136.78, 137.09, 173.03, 174.16; FAB (m/z):
391.3 [M+H]+.

EXA.MPLE 15 General procedure for dipeptide cyclization.

[004471 Diprotected dipeptides (0.545g, 1.00 mmol) were first catalytically hydrogenated in order to remove the CBZ protecting group from the N-terminal.
This was performed according to the preceding procedure, with the exception that the final recrystallization was not performed. Instead, the oil was dissolved in MeOH
(200 mL) and refluxed with DABCO (0.380g, 3 eq.) until TLC indicated completion (24 to 48 hrs).
The solution was concentrated, leaving a clear oil. Water (50mL) was added followed by dropwise addition of 1N HCI until the pH reached 2. The resulting white or light yellow precipitate was filtered, washed with water (50mL x 3), washed with ether (20mL x 3) and was dried in vacuo.

H
N D N
6k B N
A N E
F F

C
Cyclo(Trp-Trp) [00448] The three diastereomers of cyclo(Trp-Trp) of Example 15 in accordance with the present invention were as follows:

Cyclo (L-Trp-L-Trp) [00449] White solid (0.225g, 59.5%); mp 271-276 C (lit. 265-268 C); [a]DZS
141, c 0.8, MeOH (lit. -115, c 0.32, MeOH); TLC (Rf) A: 0.66, D: 0.47; IR
(vn,,,):
3413 (NH), 3328 (NH), 3056 (CH, aromatic), 2927 (CH), 1737 (N-C=0), 1671 (N-C=O), 1516 (C=C, aromatic) cm"1 ; 1 H NMR S: 2.18 (m, 2H, HA and HF), 2.70 (m, 2H, HA, and HF,), 3.88 (m, 2H, HB and HE), 6.60 (s, 2H, Ar-H), 7.00 (m, 4H, Ar-H), 7.31 (m, 4H, Ar-H), 7.72 (s, 2H, Hc and HD), 10.85 (s, 2H, indole N-H); 13C NMR:
30.87 (2C), 56.06, 56.14, 109.61 (2C), 112.12 (2C), 119.21 (2C), 119.40 (2C), 121.66 (2C), 125.26 (2C), 128.21 (2), 136.84, 136.90, 167.59 (2C); FAB (m/z): 373.3 [M+H]+.
Meso-cyclo(Trp-Trp) [00450] Light yellow solid (0.268g, 71.8%); mp 237-239; [a]D25 +1.3, c 0.8, MeOH; TLC (Rf): A: 0.64, D: 0.47; IR (vm~): 3413 (NH), 3347 (NH), 3056 (CH, aromatic), 2927 (CH), 1735 (N-C=0), 1672 (N-C=O), 1516 (C=C, aromatic) cm-';

NMR 8: 2.85 (dd, 2H, HA and HF, J=14.4 Hz, J=4.2 Hz), 3.10 (dd, 2H, HA, and HF, J=14.4 Hz, J=3.7 Hz), 3.37 (m, 2H, HB and HE), 7.0 (m, 6H, Ar-H), 7.30 (d, 2H, Ar-H, J=8.0 Hz), 7.51 (d, 2H, Ar-H, J=7.8 Hz), 7.85 (s, 2H, Hc and HD), 10.86 (s, 2H, indole N-H); 13C NMR: 29.04 (2C), 55.40 (2C), 109.14 (2C), 111.85 (2C), 119.06 (2C), 119.64 (2C), 121.53 (2C), 125.21 (2C), 128.39 (2C), 136.58 (2C), 168.37 (2C);
FAB (m/z): 373.2 [M+H]+.

Cyclo(D-Trp-D-Trp) White solid (0.241g, 62.0%); mp 276-278 C; [a]DZS +120 , c 0.8, EtOH; TLC
(Rf) A:
0.69, D: 0.49; IR (vm~): 3419 (NH), 3328 (NH), 3059 (CH, aromatic), 2927 (CH), 1735 (N-C=O), 1671 (N-C=0), 1516 (C=C, aromatic) cm"' :'H NMR B: 2.17 (m, 2H, HA and HF), 2.70 (m, 2H, HA, and HF,), 3.88 (m, 2H, HB and HE), 6.60 (s, 2H, Ar-H), 7.02 (m, 4H, Ar-H), 7.31 (m, 4H, Ar-H), 7.71 (s, 2H, Hc and HD), 10.84 (s, 2H, indole N-H);13C NMR: 30.85 (2C), 56.13 (2C), 109.61 (2C), 112.11, 112.37, 119.21 (2), 119.40 (2), 121.66 (2C), 125.26 (2C), 128.20 (2C), 136.90 (2C), 167.59 (2C).

[00451] A phase IV clinical trial of L-tryptophan (Trp) was carried out to evaluate the amino acid's effect on cognitive abilities in patients with mild to moderate Alzheimer's disease (AD). An earlier trial has been performed which studied Trp administration in patients suffering from AD [ Bentham, PW.
International Clinical Psychopharmacology, 1990, 5: 261-72]. The present trial expects the possible binding of Trp to the HHQK region of A[3 [D Giulian et al.
(1998). Journal of Biological Chemistry 273:29719-26] and subsequent disruption of A(3 plaque formation, whereas the earlier trial was based on neurotransmitter replacement therapy. By administering Trp, levels of its metabolite 5-hydroxytryptophan, better known as serotonin, were expected to increase, thereby countering deficits of the neurotransmitter experienced by those suffering from AD [
Siegel, GJ; Agranoff, BW; Albers, RW; Molinoff, PB, Basic Neurochemistry.
Fifth ed. 1994, New York: Raven Press, 1054 pp]. Also providing motivation for the trial may have been earlier accounts that Trp was seen to benefit elderly patients with mental disorders [ Shaw, DM; Tidmarsh, SF; Karajgi, BM; Sweeney, EA; Williams, S; Elameer, M; Twining, C. British Journal of Psychiatry, 1981, 139: 580-2, Lehmann, J; Persson, S; Walinder, J; Wallin, L. Acta Psychiatrica Scandinavica, 1981, 64: 123-31].

[004521 The earlier placebo-controlled Trp trial [Bentham, PW. International Clinical Psychopharmacology, 1990, 5: 261-72] suffered from several factors that hindered the generation and interpretation of data. These included a small sample size of only ten patients, two of which dropped out during the trial; problems with the sensitivity of their cognitive and behavioural tests; a relatively short observation period of three months; and the finding that their placebo group was substantially more demented at baseline. Despite these confounding features, however, the trial yielded a statistically significant correlation between the increase in Trp plasma levels and the increase in cognitive test scores. This finding, that cognition improved as a function of circulating Trp levels, was interpreted positively by the trial's lead investigator and author. In his concluding remarks, however, the author speculated that further examination of Trp's benefits in AD were unlikely to be pursued on a larger scale due to the then-recent implication of herbal supplements of Trp in the potentially fatal eosinophilia myalgia syndrome (EMS). As discussed in Section 2.3 of the protocol of Appendix A, the cases of EMS were eventually linked to a single company that produced Trp, along with an inadvertent toxic by-product, using genetically modified bacteria.

[00453] The prediction that studies using Trp would be hampered by concerns over EMS proved true, and no further clinical trials of Trp supplementation in AD
have been published in the 15 plus years since its potentially beneficial effects were identified. Recent trials, however, have shown that acute Trp depletion in patients with AD caused impairment in cognitive function [ Porter, RJ; Lunn, BS;
Walker, LL;
Gray, JM; Ballard, CG; 0 Brien, JT. American Journal of Psychiatry, 2000, 157:

40, Porter, RJ; Lunn, BS; O'Brien, JT. Psychol Med, 2003, 33: 41-9]. This finding, coupled with the discovery that AD patients have significantly reduced plasma levels of Trp [ Fekkes, D; van der Cammen, TJ; van Loon, CP; Verschoor, C; van Harskamp, F; de Koning, I; Schudel, WJ; Pepplinkhuizen, L. JNeural Transm, 1998, 105: 287-94, Widner, B; Leblhuber, F; Walli, J; Tilz, GP; Demel, U; Fuchs, D.
Adv Exp Med Biol, 1999, 467: 133-8, Widner, B; Leblhuber, F; Walli, J; Tilz, GP;
Demel, U; Fuchs, D. JNeural Transm, 2000, 107: 343-53] support a protective role for the amino acid in the aetiology of AD. The authors of the studies often suggest that a depletion of serotonin levels, resulting from reduced levels of circulating Trp, may contribute significantly to the cognitive impairment of AD [Bentham, PW.
International Clinical Psychopharmacology, 1990, 5: 261-72., Porter, RJ; Lunn, BS;
Walker, LL; Gray, JM; Ballard, CG; 0 Brien, JT. American Journal ofPsychiatry, 2000, 157: 638-40, Porter, RJ; Lunn, BS; O'Brien, JT. Psychol Med, 2003, 33:
41-9, Fekkes, D; van der Cammen, TJ; van Loon, CP; Verschoor, C; van Harskamp, F; de Koning, I; Schudel, WJ; Pepplinkhuizen, L. JNeural Transm, 1998, 105: 287-94].
Design and Progress of Trial [00454] The current trial was designed with several improvements relative to its predecessor [ Bentham, PW. International Clinical Psychopharmacology, 1990, 5:
261-72]: (1) A greater number of patients were enrolled, thus affording a larger data set for more accurate statistical analysis; (2) The cognitive tests employed, unlike those used in the earlier trial (which have since been shown to be unreliable), are widely accepted and routinely used in the fields of neurology and psychiatry (personal communication with J. Irwin, neuropsychologist); (3) The duration of the trial was six months rather than three months, increasing the likelihood of detecting a difference in the treatment group relative to the placebo group.

[00455] While Trp may benefit AD patients through a serotonergic pathway, as suggested in earlier clinical investigations [Bentham, PW. International Clinical Psychopharmacology, 1990, 5: 261-72., Porter, RJ; Lunn, BS; Walker, LL; Gray, JM;
Ballard, CG; 0 Brien, JT. American Journal of Psychiatry, 2000, 157: 638-40, Porter, RJ; Lunn, BS; O'Brien, JT. Psychol Med, 2003, 33: 41-9, Fekkes, D; van der Cammen, TJ; van Loon, CP; Verschoor, C; van Harskamp, F; de Koning, I;
Schudel, WJ; Pepplinkhuizen, L. JNeural Transm, 1998, 105: 287-94], it is also possible that Trp could bind to A(3 and inhibit fibrillogenesis. Being an "indole-anionic"

compound, Trp could form one cation-7c bond and one anionic-cationic interaction to the HHQK region of A(3. This type of binding would involve a two-point pharmacophore of Trp, comprised of its indole and carboxylate groups, binding to two of the three basic residues of the HHQK region.

[00456] Twelve patients were enrolled in the current clinical trial. Patients were administered 1 g Trp (n=8) or placebo (n=4) twice a day for six months, and their cognitive abilities were assessed, using standardized tests, at baseline, three months and six months. All enrollment and testing was carried out at the Kingston General Hospital, Kingston, Ontario, Canada, under the supervision of the principal investigator, D.F. Weaver, M.D., Ph.D (the protocol is attached as Appendix A). The primary endpoint of the double-blind trial was to determine whether patients receiving Trp outperformed the controls, either by experiencing an improvement in cognition or a reduction in deterioration in cognitive abilities relative to the placebo group.
Methods Participants [00457] Eligible participants were those who met the clinical criteria of probable Alzheimer's disease as described in the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) [ Anonymous, Diagnostic and Statistical Manual ofMental Disorders, 4th Edition (DSM-II9. 1994, Washington, D.C.:

American Psychiatric Association] and in the National Institute of Neurologic and Communicative Disorders and Stroke and the Alzheimer's Disease Related Disorders Association (NINCDS-ADRDA) [McKhann, G; Drachman, D; Folstein, M; Katzman, R; Price, D; Stadlan, EM. Neurology, 1984, 34: 939-44]. Inclusion criteria also included: mild to moderate severity of dementia, as reflected by a Mini-Mental State Examination (MMSE) [Folstein, MF; Folstein, SE; McHugh, PR. JPsychiatrRes, 1975, 12: 189-98] score of 14 to 26; minimum one-year duration of symptoms;
minimum age of 50 years; living at home or in an institution provided they had caregivers capable of attending each clinic visit and ensuring the administration of medication; able to perform the psychometric tests required; reasonably good nutritional status; vital signs (blood pressure and heart rate in sitting and standing positions), urinalysis, physical examination, and neurological evaluation must yield results within normal limits or determined as not clinically significant by the study physician for the patient's age and sex.

[00458] Patients were excluded from the trial if they had: any other cause of dementia, such as vascular dementia, as evidenced by Modified Hachinski Ischemia Scale [Rosen, WG; Terry, RD; Fuld, PA; Katzman, R; Peck, A. Ann Neurol, 1980, 7:
486-8] score greater than 4; depressive pseudementia and/or a history of more than one major depressive episode, according to DSM-IV; Creutzfeldt-Jakob disease;
clinically important head injury within one year of onset of dementia; onset of dementia following cardiac arrest or heart surgery; Huntington's chorea or Parkinson's disease, evidenced by neurological examination; DSM-IV criteria for any major psychiatric disorder including schizophrenia, alcohol or substance abuse;
history or current evidence of stroke; neurosyphilis or seropositivity for HIV; vitamin B1Z or folate deficiency; uncorrected hypothyroidism. Patients with clinically significant coexisting medical conditions including impaired renal, hepatic or gastrointestinal function were also excluded, as were patients with history or current evidence of sleep disorder.

[00459] The following medications were prohibited for safety reasons or to prevent a false positive result: investigational drugs for AD or for any other disorder, MAO inhibitors (e.g. phenelzine, tranylcypromine, noclobemide), SSRI
antidepressants (e.g. fluoxetine, paroxetine), lithium, acetyl-cholinesterase inhibitors (e.g. donepezil, galantamine), ginkgo biloba, ginseng, vitamin E
supplementation, estrogen for more than hormonal replacement, aspirin at doses greater than 650 mg/day, non-steroidal anti-inflammatory drugs (NSAIDS, e.g. ibuprofen, naproxen, diclofenac) for more than 30 consecutive days (allowed as required).
Information on allowed concomitant treatments taken by patients was recorded at each visit.

[00460] Written informed consent was obtained from patients and their substitute decision-makers before the initiation of any testing procedures.
The study was reviewed and approved by the Queen's University Health Sciences and Affiliated Teaching Hospitals Research Ethics Board, Kingston, Canada.

Study Design [00461] This study was a 6-month, double-blind randomized trial in which eligible participants were randomly assigned to receive either Trp (1000 mg, twice daily) or an identical-appearing placebo. Recruiters were unaware of the assignment.
The randomization ratio was two to one for Trp versus placebo, as has been used previously in AD clinical trials [Erkinjuntti, T; Kurz, A; Gauthier, S;
Bullock, R;
Lilienfeld, S; Damaraju, CV. Lancet, 2002, 359: 1283-90, Erkinjuntti, T; Kurz, A;
Small, GW; Bullock, R; Lilienfeld, S; Damaraju, CV. Clin Ther, 2003, 25: 1765-82].
Doses consisted of a single capsule and were administered one hour before breakfast and at bed time each day. The medication was taken without food to prevent absorption competition from dietary amino acids such as phenylalanine [Kilberg, M;
Haussinger, D, Mammalian Amino Acid Transport Mechanisms and Control. 1992, Plenum: New York. p. 166-7]. A dosage diary was filled out by the participant's caregiver to ensure compliance. After the initial screening, clinic visits took place at 0, 3 and 6 months. Blinding was maintained until all patients completed the trial.
[00462] The primary efficacy measures were the change in MMSE [Folstein, MF; Folstein, SE; McHugh, PR. JPsychiatr Res, 1975, 12: 189-98] and Alzheimer's Disease Assessment Scale, cognitive subpart (ADAS-Cog) [Rosen, WG; Mohs, RC;
Davis, KL. Am JPsychiatry, 1984, 141: 1356-64]. The MMSE is a short standard assessment for diagnosing the presence and severity of cognitive impairment, and evaluates six domains of cognitive functioning: orientation, registration, attention, recall, language and constructional abilities. The maximum score is 30 points, with lower scores indicating a greater degree of cognitive impairment. The ADAS-Cog is an 11-item test battery that relies solely on the patient's ability to perform specific tasks during the administration of the test. It ranges from 0 to 70 points, with higher scores indicating a greater degree of cognitive impairment.

[00463] Secondary efficacy measures were the Alzheimer's Disease Cooperative Study - Clinical Global Impression of Change (ADCS-CGIC) [Schneider, LS; Olin, JT; Doody, RS; Clark, CM; Morris, JC; Reisberg, B;
Schmitt, FA; Grundman, M; Thomas, RG; Ferris, SH. Alzheimer Dis Assoc Disord, 1997, 11 Suppl 2: S22-32], a rating designed to describe the change, as determined by the clinician, occurring in a patient from baseline; Neuropsychiatric Inventory (NPI) [Cummings, JL; Mega, M; Gray, K; Rosenberg-Thompson, S; Carusi, DA; Gombein, J. Neurology, 1994, 44: 2308-14], a test performed by a neuropsychologist to assess ten behavioural disturbances that can occur in patients with dementia:
delusions, hallucinations, agitation/aggression, dysphoria, anxiety, elation/euphoria, apathy, disinhibition, irritability/lability and aberrant motor behaviour; Disability Assessment for Dementia (DAD) [Gelinas, I; Gauthier, L; McIntyre, M; Gauthier, S. Am J
Occup Ther, 1999, 53: 471-81 ], a test used to quantitatively measure functional abilities in the activities of daily living, such as meal preparation and housework, in individuals with cognitive impairments; Functional Activities Questionnaire (FAQ) [Pfeffer, RI;
Kurosaki, TT; Harrah, CH, Jr.; Chance, JM; Filos, S. J Gerontol, 1982, 37: 323-9], a caregiver-based measure of functional activities; and a clock-drawing test [Tuokko, H; Hadjistavropoulos, T; Miller, JA; Beattie, BL. JAm Geriatr Soc, 1992, 40:

84], an instrument that assesses a range of cognitive abilities, including executive functioning and visuospatial perception. At each visit after baseline, participants were also questioned about any adverse events experienced since beginning the trial.
[00464] Since Trp or its metabolites may have sedative [Wyatt, RJ; Engelman, K; Kupfer, DJ; Fram, DH; Sjoerdsma, A; Snyder, F. Lancet, 1970, 2: 842-6, Mannaioni, G; Carpenedo, R; Corradetti, R; Carla, V; Venturini, I; Baraldi, M;
Zeneroli, ML; Moroni, F. Adv Exp Med Biol, 1999, 467: 155-67] or anti-depressant properties [Boman, B. Aust N ZJPsychiatry, 1988, 22: 83-97], the Pittsburgh Sleep Quality Index [Buysse, DJ; Reynolds, CF, 3rd; Monk, TH; Berman, SR; Kupfer, DJ.
Psychiatry Res, 1989, 28: 193-213] and Cornell Depression Index [ Alexopoulos, GS;
Abrams, RC; Young, RC; Shamoian, CA. Biol Psychiatry, 1988, 23: 271-84] were performed to ensure that any change in a patient's dementia is unrelated to these potential effects.

[00465] Primary efficacy variables, the Pittsburgh Sleep Quality Index and Cornell Depression Index were measured at 0, 3 and 6 months, while secondary efficacy variables were measured at 0 and 6 months.

Statistical Analyses [00466] All statistical comparisons were two-tailed, with p values < 0.05 considered significant. Analysis was based on intention to treat, comparing mean change from baseline in primary and secondary efficacy measures between treatment and control groups by means of t-tests. All efficacy measures found significantly associated with treatment status were entered into an ANOVA model with baseline score and treatment status as independent variables and change in score as the dependent variable. Baseline characteristics were tested for significant differences between treatment and control groups by t-test (for continuous variables) and xZ for categorical variables. Within the treatment group, change in scores from baseline of those primary and secondary outcome efficacy measures found to be significantly associated with treatment status were tested for significance by means of two-tailed paired t-tests.

Results [00467] The treatment and placebo groups had similar characteristics at baseline (Table 5). Although the treatment group consistently trended towards higher baseline scores on the cognitive tests, none of the differences reached significance.

Table S. Baseline characteristics Characteristic Placebo Trp P
n= 4 n= 8 value Demo ra hics Women (%) 2 (50) 4 (50) 1.0 Age, mean (SD) 72.8 71.4 0.80 9.1 (8.3) Weight, mean (SD), kg 64.5 70.0 0.58 (5.8) (18.2) Cognitive function, mean score (SD) MMSE 21.0 22.4 0.61 (3.4) (4.7) ADAS-Cog 22.8 20.4 0.65 (7.2) 8.7) DAD 67.8 84.5 0.27 (26.5) (22.2) FAQ 18.8 10.9 0.18 (6.7) (9.8) NPI 15.5 17.8 0.78 (8.7) (13.9) Clock-drawing 3.8(4.2) 6.2 (3.0) 0.26 [00468] No adverse events were reported by patients taking either Trp or placebo, and no patients withdrew from the trial. All dosage diaries were returned indicating complete compliance. All patients entered the intention-to-treat analysis.
[00469] Patients assigned Trp improved in cognitive function relative to patients receiving placebo (Table 6, Figure 15). In terms of the primary efficacy variables, the treatment group had a significantly improved change in MMSE
score at 3 months (p=0.009) and 6 months (p<0.001), while the improvement in ADAS-Cog relative to controls was significant at 6 months (p=0.017), but not at 3 months (p=0.38). Improvements were also seen after 6 months on two of the five secondary efficacy measures: ADCS-CGIC scores were higher and change in clock test was improved in patients receiving Trp relative to the placebo group (Table 6).
All significant improvements on primary and secondary efficacy measures remained significant when baseline test score was controlled for (data not shown).

Table 6. Efficacy outcomes after 3 and 6 months Outcome Placebo (n=4) Trp (n=8) Treatment P value measure difference Tr vs. placebo) Mean SE change from baseline after 3 months MMSEa -1.2 (0.5 0.8 (0.4) 2.0 0.009 ADAS-Cog 0.5 (0.3) -0.8 (0.9) 1.3 0.39 Mean (SE) chan e from baseline after 6 months MMSEa -3.2 (0.6) 1.8 0.5 5.0 <0.001 ADAS-Cog 3.5 (1.0) -2.8 (1.4) -6.3 0.017 DADa -11.2 (9.0) 1.5(1.8) 12.8 0.25 FAQ 5.2 (1.4 3.2(2.0) -2.0 0.53 NPI 22(18) 3 (3) -19 0.38 Clock-drawinga -1.8 (0.3) 1.1 (0.4) 2.9 <0.001 ADCS-CGIC' 6.5 (0.3) 3.6(0.3). 2.9 <0.001 a Positive change indicates improvement.
b Negative change indicates improvement.
Score at 6 months, not change from baseline (score is a measure of change).
Lower score indicates improvement.

[00470] At 6 months, improvement in the treatment group from baseline was significant for MMSE (p=0.006) and clock test (p=0.015), though not for ADAS-Cog (p=0.097). The improvement in MMSE score at 3 months in people taking Trp likewise failed to reach significance (p=0.08).

[00471] No difference was found to exist in the moods of patients in the treatment versus the placebo group; no patients were depressed at baseline, as indicated by a Cornell Depression Index score of> 5, while two patients taking Trp and one taking placebo were depressed after 6 months of treatment (i.e. 25% of each group). No sleep problems, identified by a score of> 4 on the Pittsburgh Sleep Quality, were reported at baseline or at 6 months in either the treatment or placebo groups.

Discussion [00472] This trial found that administration of Trp was of benefit to individuals with Alzheimer's disease. Cognitive abilities, measured by the MMSE and ADAS-Cog tests, improved in patients taking Trp, both relative to baseline and to patients receiving placebo.

[00473] The trial suffered from many limitations, particularly small size.
While it was originally planned to enroll 30 patients in the trial (20 treatment, 10 placebo), limitations in the enrollment period only allowed for the recruitment of 12 patients. A larger cohort may have lead to smaller differences on baseline cognitive tests between the two groups; although none of the differences were found to be significant, there was a consistent trend towards greater cognitive abilities in the treatment group. Since people at more advanced stages of AD are known to decline faster [Mitnitski, AB; Graham, JE; Mogilner, AJ; Rockwood, K. J Gerontol A
Biol Sci Med Sci, 1999, 54: M65-9], this trend may account for some of the difference in cognitive outcomes between the two groups.

[00474] No difference was seen to exist between the placebo and treatment groups in terms of the number of people reporting sleep disturbances or depression, either at baseline or at completion of the trial. Since both are binary measures, however, moderate improvements or declines in sleep quality or mood could not be detected. If such discrepancies existed between the treatment and control groups, they could have had an effect on the measured efficacy of Trp.

[00475] In the earlier trial of Trp in patients with AD [ Bentham, PW.
International Clinical Psychopharmacology, 1990, 5: 261-72], no difference in depression, measured by the Montgomery and Asberg Depression Rating Scale [Montgomery, SA; Asberg, M. BrJPsychiatry, 1979, 134: 382-9], was found between treatment and placebo groups. Data on sleep quality were incomplete and therefore not analyzed.

[00476] Not only did all the primary and secondary efficacy tests trend towards, or were found significantly associated with, the treatment group outperforming the placebo group, but five of the seven tests (all but FAQ, NPI) suggested an increase in cognitive abilities over the course of the trial for those receiving Trp. Two of these efficacy measures (MMSE and clock test) were found to be significantly improved at 6 months relative to baseline.

[00477] The efficacy measures in which Trp showed a significant benefit (MMSE, ADAS-Cog, clock test and CGIC) all evaluate higher-level cognitive function, while the tests that showed no benefit from Trp (FAQ, DAD and NPI) evaluate behavioural disturbances and functional abilities. While it may be that Trp acts to improve cognitive but not non-cognitive faculties, other explanations may account for this finding. Measuring behavioural disturbances, for instance, is inherently associated with greater uncertainty than evaluating specific cognitive skills such as arithmetic or word recall. The greater uncertainty in measuring non-cognitive functioning may have reduced the ability to detect differences between the treatment and placebo groups in the FAQ, DAD and NPI tests, especially in a study with such low power.

[004781 This is not the first account of Trp benefiting patients with dementia.
In the only prior randomized clinical trial in patients with AD [Bentham, PW.
International Clinical Psychopharmacology, 1990, 5: 261-72], the author found a significant correlation between increase in Trp plasma levels and increase in scores on the Information, Memory, Concentration test [Blessed, G; Tomlinson, BE; Roth, M.
BrJPsychiatry, 1968, 114: 797-811]. Several other efficacy measures used in the trial trended towards an improvement in the treatment group relative to the placebo group. In an earlier uncontrolled study, Shaw et al. found an "unequivocal improvement in blind global assessment" in six of 29 patients with advanced senile dementia, though not specifically AD, after daily Trp administration (24 mg/kg) [Shaw, DM; Tidmarsh, SF; Karajgi, BM; Sweeney, EA; Williams, S; Elameer, M;
Twining, C. British Journal of Psychiatry, 1981, 139: 580-2]. None of the patients taking placebo showed improvement.

[00479] Conversely, a double-blind crossover trial of Trp in patients with dementia showed the drug to be of no benefit [Smith, DF; Stromgren, E;
Petersen, HN; Williams, DG; Sheldon, W. Acta Psychiatr Scand, 1984, 70: 470-7]. It is possible that differences in trial design account for the lack of effect:
patients were all residing in a nursing home; the duration of the trial was only one month;
patients with any form of dementia, rather than specifically AD, were enrolled in the study, including some whose dementia was very advanced; dosing was only performed once daily; Trp was co-administered with a high-protein food (yogurt or chocolate milk);
and many patients suffered from depression. Of particular interest, however, the efficacy measures used were nurse [Plutchik, R; Conte, H; Lieberman, M; Bakur, M;
Grossman, J; Lehrman, N. JAm Geriatr Soc, 1970, 18: 491-500] and psychologist [Gotestam, KG. Acta Psychiatr Scand Suppl, 1981, 294: 54-63] ratings of patients' behaviour and functional abilities. As mentioned above, Trp was found in this trial to be ineffective at improving these domains, as reflected by scores on the FAQ, DAD
and NPI tests.

[00480] The mechanism of action of Trp in AD has been discussed. Authors of earlier trials [Bentham, PW. International Clinical Psychopharmacology, 1990, 5:
261-72, Porter, RJ; Lunn, BS; Walker, LL; Gray, JM; Ballard, CG; 0 Brien, JT.

American Journal ofPsychiatry, 2000, 157: 63 8-40, Porter, RJ; Lunn, BS;
O'Brien, JT. Psychol Med, 2003, 33: 41-9, Fekkes, D; van der Cammen, TJ; van Loon, CP;
Versehoor, C; van Harskamp, F; de Koning, I; Schudel, WJ; Pepplinkhuizen, L. J
Neural Transm, 1998, 105: 287-94] proposed the drug could act through a serotonergic pathway, i.e. increased Trp intake would lead to greater serotonin synthesis. However, while clinical trials of selective serotonin reuptake inhibitors (SSRIs) in AD [Lyketsos, CG; DelCampo, L; Steinberg, M; Miles, Q; Steele, CD;
Munro, C; Baker, AS; Sheppard, JM; Frangakis, C; Brandt, J; Rabins, PV. Arch Gen Psychiatry, 2003, 60: 737-46, Teri, L; Logsdon, RG; Peskind, E; Raskind, M;
Weiner, MF; Tractenberg, RE; Foster, NL; Schneider, LS; Sano, M; Whitehouse, P;
Tariot, P;
Mellow, AM; Auchus, AP; Grundman, M; Thomas, RG; Schafer, K; Thal, LJ.
Neurology, 2000, 55: 1271-8, Nyth, AL; Gottfries, CG. BrJPsychiatry, 1990, 157:
894-901, Olafsson, K; Jorgensen, S; Jensen, HV; Bille, A; Arup, P; Andersen, J. Acta Psychiatr Scand, 1992, 85: 453-6] have shown improvement in a number of noncognitive symptoms, they have shown no effect on cognition. Furthermore, neither this trial, nor its predecessor [Bentham, PW. International Clinical Psychopharmacology, 1990, 5: 261-72] found a difference in depression between the treatment and placebo groups. These findings suggest a non-serotonergic mechanism of action for Trp's activity in AD.

[00481] As a non-toxic, readily-absorbed and naturally abundant compound, it is possible that Trp has the potential to be a future treatment for AD.

[00482] Binding energies of L-Trp to the HHQK region of A[3 were calculated using the CHARM27 force field and explicit solvation (Table 7). Using CHARMM22, an earlier version of CHARMM, the binding energies of L-Trp to PDB structures of A(3 were similar to those for sodium 1,3-propanedisulfonate, a known Ap anti-aggregant (Kisilevsky et al., Nature Medicine, 1:143-148, 1995).

Table 7 [00483] Binding energies of L-Trp to HHQK region of Ap using the CHARMM27 force field and explicit solvation.

PDB Structure L-Tr Binding at:
His13-His14 His13-L s16 1 AMB -54.4 -43.5 IAMC -44.6 -34.3 IAML -35.6 -22.3 1 BA4 -27.1 -46 IIYT -36.8 -17.3 2BP4 -32.7 -48.6 Notes:

Energies are in kcal/mol.

CHARMM27 force field is a molecular mechanics computer program.

Ref.: A. D. MacKerell et al., Journal of Physical Chemistry B, 102:3586-3616, 1998.
Explicit solvation means water molecules were present during the binding of L-Trp to AR.

PDB = Brookhaven Protein Databank [HM Berman et al. (2000). Nucleic Acid Research 28:235-42].

These 6 different PDB files are of various lengths of A[l, and were generated from NMR experiments under different conditions (pH, solvent, temp). Two different binding motifs of L-Trp to the HHQK region of A(3 are shown in Figures 14 and 15.

Figure 14 gives the "typical" interaction found for binding at His13 and Lys16 of A[3, here to PDB structure 1AML. Significant binding interactions at His13 and HisJ4 were also found. Figure 15 is an alternative interaction found at residues His14 and Lys16 of the PDB structure 1BA4. Asp,, G1y9 and Va1]2 also participate in binding.

Molecular Minimization Results [00485] It has been suggested that Alzheimer's Disease (AD) is a multifactorial condition, with a diverse group of proteins implicated in its pathogenesis.
Stephenson et. al. (2005. FEBS Lett 579:1338-42) have identified a common BBXB motif present in the peptide sequences of 27 such AD associated proteins. It is from this group of BBXB-containing proteins (BCPs) that several were selected for a series of in silico calculations with the objective of determining the ability of a novel molecule, Oc, to bind to their BBXB receptors and potentially neutralize their action and toxicity. Figure 16 depicts the interaction of Oc with KREH receptor of B7-1.
Figure 17 depicts the interaction of Oc with RDHH receptor of ICAM-1. Figure 18 depicts interaction of Oc with HKEK receptor of IL-IR1.

Methods [00486] A collection of molecular systems were constructed, each consisting of a three dimensional structure of a BCP, obtained from the ExPASy Protein Knowledgebase (Apweiler R et al. 2004. Nucleic Acids Research 32:115-9), with a manually modelled molecule of Oc placed in close proximity to a BBXB receptor of said protein. Subsequently, these systems were each minimized using the Chemical Computing Group Molecular Operating Environment (MOE) software (The Chemical Computing Group, Montreal, Canada, 2000), and the resulting bonding energy and separation distance of Oc to BBXB receptor interaction calculated. These values were then compared to separation distance and bonding energy values calculated for a control system consisting of an interaction between the HQHK receptor of IL-and pentane, a molecule not expected to have strong interactions at BBXB
receptors.
Representative interactions between Oc and some BCPs are found in Figures 16, and 18.

Table 8. Results of in silico simulations between Oc and a series of BBXB
receptors found in various proteins believed to be associated in the pathogenesis of Alzheimer's Disease.

Substrate: OC
Protein PDB File Target Receptor Binding Energy Approximate Separation ~kcal/mol) (A) B7-1 1 DR9 KREHb -30.6 2 BHMT 1 LT7 RARKa -5.7 8 RARe -17.4 3 C1qA 1PK6 KKGH -12.3 2 HFE 1A6Z HKIR -1.0 6 ICAM-1 11AM RDHHb -30.5 3 RRDH -12.9 3 IFN-g IEKU KKKRa -8.8 2 KKKReb -24.3 3 IL-12B 1F42 KSKR -0.4 6 HKLKb -15.9 2 IL-13 1 GA3 l-{LKKb -18.6 3 IL-4 IBBN HHEKb -15.5 3 IL-1 R1 1ITB HKEKb -43.8 3 HQHKb -17.0 2 MIP-la 1B53 KRSRb -30.7 3 MIP-1R 1HUM KRSKe -6.7 2 KRSKab -16.1 2 Neprilysin 1 D19 HCRKb -28.8 3 KKCRb -44.5 3 KKLRb -35.9 3 S100R 1 UWO HKLKb -34.8 3 KLKK -13.3 8 SDF-1 2SDF KHLK -10.5 3 Transferrin 1N84 RGKKb -18.7 3 A,-ACT 1AS4 KRWRb -25.8 3 Control Substrate: pentane Binding Energy Approximate Separation Protein Target Receptor (kcal/mol) (A) IL-1 R1 1 ITB HKEK -9.8 3 e Multiple occurrences of sequence in PDB file studied b Denotes favourable interaction with Oc, defined as <-15 kcal/mol Table 9. Parameters passed to MOE for purposes of molecular minimization of sample systems.

Initial Refinement Parameters Force Field CHARMM27 Gradient 0.05 Partial Charge Calculation Enabled Atom Tethering Enabled Backbone Bonded Enabled van der Waals Enabled Electrostatics Enabled Restraints Enabled Cutoff Enabled On: 8 Off:10 Solvation Distance Die[ectric:1 Exterior: 80 Scale Like:1 Unlike: 0 Wild:1 Final Refinement Parameters Force Field CHARMM27 Gradient 1 Partial Charge Calculation Enabled Atom Tethering N/A
Bonded Enabled van der Waals Enabled Electrostatics Enabled Restraints Enabled Cutoff Enabled On: 8 Off:10 Solvation Gas Phase Dielectric: I Exterior: 80 Scale Like:1 Unlike: 0 Wild: 1 Water Soak Solute: All Mode: Box Width: 5 [00487] Minimizations were carried out using the CHARMM27 force field (Brooks BR et al. 1983. J Comput Chem 4:187-217; MacKereil AD Jr. 1998.
CHARMM: The Energy Function and Its Parameterization with an Overview of the Program. In Encyclopedia of Computational Chemistry, Schleyer, PR et al., eds., John Wiley & Sons, Chichester, Vol. 1, pp 271-277), and consisted of a two-step process (Table 9). Initially, an optimization was conducted on the system without solvent molecules, so that the binding geometries could be refined, producing a substrate that was oriented in a chemically realistic fashion. The refined system was then explicitly solvated with water molecules in order to simulate physiological circumstances, and again optimized to further improve system geometries. The resulting geometries, which better represent molecular behaviour in vivo, were then analysed, and binding energies and separation distances were calculated (Table 9). A favourable interaction was defined as one in which the binding energy of Oc to the BBXB receptor was greater than 15 kcal/mol (i.e. the system energy was <-15 kcal/mol), with a substrate to receptor separation distance of less than or equal to 3A. Using these criteria, favorable interactions were found to exist between Oc and 17 of the 26 BCP
systems analyzed. The pentane-to-HQHK control interaction was only found to have a binding energy of -9.8 kcal/mol.

1004881 The in silico simulations performed suggest that Oc is able to bind to the BBXB motifs of a number of proteins involved in AD pathogenesis, and that the compound and ones with similar structures may therefore be beneficial in treating AD.

Molecular modeling study: aromatic groups for cation-ir binding to HHQK
1004891 A study was carried out that examined, via ab initio methods, the interaction of methylammonium (MA), the end of lysine's sidechain, and 4-methylimidazolium (4-MI), the protonated sidechain of histidine, with a series of 1 1 monocylic and bicyclic aromatic species (benzene, pyridine, pyrrole, thiophene, furan, naphthalene, indole, quinoline, isoquinoline, benzothiophene and benzofuran).

The goal of the study was to evaluate, using Gaussian98 (Revision A.9. 1998, Gaussian Inc., Pittsburgh, PA, U.S.A.), the ability of different aromatic systems to bind to one of the histidine or lysine residues in the HHQK region of A[3.
Methods [00490] MA and 4-MI and each aromatic group studied were individually geometry-optimized at the restricted Hartree-Fock (RHF) level using the 3-21G
basis set in the Gaussian98 computer program. Once optimized, the single-point RHF
and MP2 energies of the binding complexes and those of the cations and the aromatic species on their own were calculated using the 6-31 G(d) basis set. Binding energies were determined by subtracting the sum of the monomer energies from the energy of each complex.

Results [00491] The trend in binding strength at the MP2/6-31G(d) level, using the most energetically favourable interaction for each aromatic, be it charge-dipole or cation-7c in nature, is shown in Table 10. Pyridine and pyridine-containing aromatic groups (quinoline and isoquinoline) formed the strongest interactions, though these were charge-dipole in nature, and would therefore be expected to be of much smaller magnitude in an aqueous environment (i.e. in vivo). Indole formed the strongest cation-TE interactions, followed by naphthalene and pyrrole. The magnitude of these may be equal to or greater than the charge-dipole interactions formed by pyridine, quinoline and isoquinoline when solvation is taken into account.

[00492] Accordingly, indole was a preferred aromatic group incorporated into certain compounds employed in methods of the invention.
A

E
i 25 'a'~ //~S
' qJ
~ ~a ctio ~

Table 10. Binding of MA and 4-MI to aromatic systems. MP2/6-31 G(d)//RHF/3-21 G
binding energies of MA and 4-MI to the aromatic systems studied. Only the most energetically favourable binding complex is included.

[00493] Many other variations of the present invention will be apparent to those skilled in the art and are meant to be within the scope of the claims appended hereto.

A12pendix A: Protocol for Clinical Trial DOUBLE-BLIND, MEDIUM TERM EFFICACY STUDY OF TRP01 IN PATIENTS WITH MILD TO MODERATE ALZHEIMER'S DISEASE
Study Investigator and Manager Neuropsycholmist Dr. Donald Weaver Dr. Jill Irwin Kingston General Hospital Kingston General Hospital 76 Stuart St., 76 Stuart St., Kingston, Ontario, K7L 2 V7 Kingston, Ontario, K7L 2V7 Nurse Coordinator Research Assistant Ms. Sandy Weatherby Mr. Michael Carter Kingston General Hospital Rm. 505, Frost Building 76 Stuart St., Queen's University, Kingston, Ontario, K7L2V7 Kingston, Ontario, K7L 3M6 CONFIDENTIAL
TABLE OF CONTENTS

1. INTRODUCTION 178 1.1 Alzheimer's disease (AD) 178 1.2 Causes of the onset of AD 178 1.3 Motivation for use of TRPOI in the treatment of AD 178 2. TRYPTOPHAN 179 2.1 Pharmacology 179 2.2 Tolerance 180 2.3 Toxicity 180 2.4 Drug interactions 181 2.5 Safety 181 3. STUDY OBJECTIVE 182 4. STUDY DESIGN 182 5. SELECTION OF STUDY POPULATION 182 5.1 Inclusion criteria 182 5.2 Exclusion criteria 183 5.3 Recruitment 185 5.4 Number of subjects 185 6. STUDY DRUGS 185 6.1 Study medication 185 6.2 Drug supplies and accountability 185 6.3 Assignment of treatments to patients 186 6.4 Concomitant treatments 186 7. EVALUATION 187 7.1 Evaluation criteria 187 7.1.1 Tolerance criteria 187 7.1.2 Efficacy criteria 187 7.1.3 Compliance criteria 188 7.1.4 Other parameters 188 7.2 Methods of evaluation 188 7.2.1 Efficacy 188 7.2.2 Tolerance and safety 190 7.2.3 Compliance 190 7.2.4 Other parameters 191 8. STUDY PROCEDURE 191 8.1 Pre-treatment 191 8.2 Treatment 192 8.3 Post treatment 192 9. ADVERSE EVENTS 192 9.1 Definition 192 9.2 Period of observation 193 9.3 Procedures to be followed 193 9.4 Specific case of serious adverse events 193 10. WITHDRAWALS AND DEFAULTERS 194 11. COLLECTION AND ANALYSIS OF DATA 195 11.1 Case report forms 195 11.2 Number of patients 195 11.3 Statistical methods 195 11.3.1 Data handling procedures 195 11.3.2 Statistical analysis 195 12. ETHICAL AND LEGAL ASPECTS 196 12.1 Declaration of Helsinki 196 12.2 Patient information and consent 197 12.3 Ethics committee approval / information 197 12.4 Confidentiality 197 13. PROTOCOL AMENDMENTS - 197 PREMATURE DISCONTINUATION OF STUDY
13.1 Protocol amendments 197 13.2 Premature discontinuation 198 14. USE OF INFORMATION AND PUBLICATION 198 15. TIME TABLE 198 16. DECLARATION OF TRIAL PERSONNEL 199 17. DECLARATION OF INVESTIGATOR 200 18. REFERENCES 201 PROTOCOL OUTLINE

TITLE Double-blind, medium-term efficacy study of TPRO1 in patients with mild to moderate Alzheimer's Disease STUDY SITE Kingston General Hospital, Kingston, Ontario, Canada PHASE IV

INDICATION Mild to moderate Alzheimer's Disease (AD) STUDY OBJECTIVE To evaluate early signs of any interruption in the progressive deterioration of cognitive abilities in patients with AD.

MEDICATION/ Group A: II 1g BID for 26 weeks DOSAGE Group B: placebo lg BID for 26 weeks DESIGN Affter an initial set of screening tests, patients will be randomized into either Group A(TRP01) or Group B
(placebo). Patients will take their medication in a double-blind manner for 26 weeks.

POPULATION Male and female patients, at least 50 years of age, with a diagnosis of dementia (DSM-IV criteria) and probable Alzheimer's Disease (NINCDS-ADRDA criteria) of mild to moderate severity, i.e. a Mini-Mental State Examination score of 14 to 26 inclusive.

SAMPLE SIZE 30 patients EFFICACY MMSE, ADAS-Cog, ADCS-CGIC, NPI, DAD, FAQ, VARIABLES Clock test SAFETY VARIABLES Physical and neurological examinations, vital signs, adverse events recording, urinalysis TIMETABLE First patient screened: April 2001.
End of study: recruitment will be stopped after achievement of the planned number of patients. The last patient will complete treatment by January 2002.
STUDY FLOW CHART

Visit 0* 1* 2 3 Weeks / -4 to 0 0 13 26 Start of treatment Written informed consent X
Full medical history X
Physical and neurological X X
examinations Weight X X
Vital signs X X X
Urinalysis X X
Concomitant medications X X X X
Hachinski scale X
Cornell Depression Index X X X
Pittsburgh Sleep Quality Index X X X
MMSE X X X X
ADAS-Cog X X
Clock test X X X
ADCS-CGIC X X
NPI X X
DAD X X
FAQ X X
Adverse events X X

* Visits 0 and 1 can be combined if patient does not require a 4 week wash-out period for prohibited medication (see section 6.4) DESIGN SCHEMATIC

Optional continuation E- Screening ~: 1g TRP01 or 1g placebo BID-> p tient's expense i i 1 --~
---- - -Visit 0 Visit 1 Visit 2 Visit 3 Week -4 Week 0 Week 13 Week 26 1. INTRODUCTION

1.1 Alzheimer's Disease Alzheimer's Disease (AD) is a brain disorder characterized by a progressive dementia that occurs in middle or late life and which affects 5% of people over 65 years of age. The pathological characteristics are degeneration of specific nerve cells, presence of neuritic plaques, and neurofibrillary tangles.
Alterations in transmitter-specific markers include forebrain cholinergic systems and, in some cases, noradrenergic and somatostatinergic systems that innervate the telencephalonI .

AD causes dementia, the essential feature of which is the development of multiple cognitive deficits that include memory impairment and at least one of the following cognitive disturbances: aphasia, apraxia, agnosia or a disturbance of executive functioning. Although AD is a common cause of dementia, other causes exist, including Parkinson's Disease, head trauma, Huntington's disease and HIV disease2.

1.2 Causes of the onset of AD
The discovery of extracellular co-deposits of (3-amyloid, a peptide of 39 to amino acids, and polysulfated glycosaminoglycans (GAGs) in the brains of patients with AD has motivated the theory that these aggregates cause the onset of the disease3. The neurotoxicity of (3-amyloid to neuronal cells has been demonstrated4, thus adding support to the theory.

1.3 Motivation for use of TRPO1 in the treatment of AD
Inhibition of (3-sheet fibril formation has been shown to occur in vitro in the presence of small molecules incorporating sulphate or sulphonate functional groupss. These small molecules are believed to mimic endogenous GAGs and thereby interrupt the (3-amyloid/GAG association required for formation of amyloid deposits in vivo.

1 McKhann G; Drachman D; Folstein M; Katzman R; Price D; Stadlan EM.
Neurology (1984) 34: 939-44.

2 Baldereschi M; Amato MP; Nencini P; Pracucci G; Lippi A; Amaducci L;
Gauthier S; Beatty L; Quiroga P; Klassen G. Neurology (1994) 44: 239-42.
3 Varghese J; Latimer LH; Tung JS; Dappen MS. Ann. Rep. Med. Chem.
(1997) 32:11-19.

4 Simmons LK; May PC; Tomaselli KJ; Rydel RE; Fuson KS; Brigham EF;
Wright S; Lieberburg I; Becker GW; Brems DN; Li WY. Mol. Pharmacology (1994) 45: 373-79.

Kisilevsky R; Lemieux LJ; Fraser PE; Kong X; Hultin PG; Szarek WA.
Nature Medicine (1995) 1: 143-148.

Although the sulfate/sulfonate groups were effective at in vitro inhibition of fibril formation, their negatively-charged functional groups render the molecules incapable of crossing the blood-brain barrier6. This generates the need for a molecule which can duplicate the success of the sulfate/sulfonate goups at inhibiting (3-fibril formation but which can also pass through the blood-brain barrier. L-tryptophan (TRPOI) is known7 to meet the latter of these criteria and is expected to meet the former for reasons outlined below.
GAGs and their sulfated/sulfonated mimics are thought to bind to positively-charged residues on the R-am&id peptide through cationic/anionic interactionsa. Kinetic studies have revealed that these positively-charged residues must reside in the central region of the (3-amyloid peptide. In other studies1,10, the His13-His14-G1n15-Lys16 region has been specifically identified as the most likely point of GAG/(3-amyloid interaction.

In light of this information, it is necessary to consider the ability of TRP01 to interact with this sequence in order to assess its potential for inhibiting aggregation. It has been shown" through examination of 3D protein x-ray crystal structures, that TRP01 and His residues are often in close proximity, suggesting an affinity for one another. Such an affinity has been attributed to a combination of hydrophilic and hydrophobic effects by some" while other evidence suggest that aromatic-cationic interactions, also termed cation-zc interactions, are responsib1e12. Irrespective of the mechanism, the data from the protein crystal structures" suggest that TRPO1 could possibly interact with one or both of His13 and His14 on the (3-amyloid.

In the study of x-ray crystal structures mentioned above, it was also shown that TRPO 1 residues are found close to Lys residues with a frequency statistically greater than random. This is likely due to the formation of cation-zt interactions known to occur between the sidechains of these amino acids12 .
6 Pardridge WM. Advanced Drug Delivery Reviews (1995) 15: 3-36.

' Kilberg MS; Haussinger D. Mammalian Amino Acid Transport Mechanisms and Control, Plenum, New York, 1992; p.166-7.

$ Brunden KR; Richter-Cook NJ; Chaturvedi N; Frederickson RCA. J.
Neurochem. (1993) 61: 2147-2154.

9 Snyder SW; Ladror US; Wade WS; Wang GT; Barrett LW; Matayoshi ED;
Huffaker HJ; Kraft GA; Holzman TF. Biophysical Journal (1994) 67: 1216-1228.

Soto C; Branes MC; Alvarez J; Inestrosa NC. J. Neurochem. (1994) 63:
1191-1198.

" Karlin S; Zuker M; Brocchieri L. J. Mol. Biol. (1994) 239: 227-248.

12 Gallivan JP; Dougherty DA. Proc. Natl. Acad. Sci. USA (1999) 96: 9459-64.

The results from this study suggest that TR.P01 could potentially interact with Lys16 of (3-amyloid.

Since TRPO1 appears to have the ability to interact with both His and Lys residues, it is possible that it will interfere with (3-amyloid deposition in vivo by interacting with the amyloid's His13-His14-Gln15-Lys16 region. This would prevent (3-amyloid from interacting and co-precipitating with GAGs, thereby inhibiting any further build-up of neuronal plaques in patients with AD.
Coupling this expectation with TRPO1's proven ability to cross the blood-brain barrier recommends TRP01 as a potential therapeutic agent in the treatment of AD.

2. TRYPTOPHAN13 2.1 Pharmacology L-tryptophan is 1 of the 8 essential amino acids and has a recommended minimum daily intake of 0.25 g for males, 0.15 g for females. It is present in the hydrolysates of most proteins with the average westem diet containing 1 to 3 grams per day. There are two main metabolic pathways for TRPOl;
approximately 98% of dietary TRPO l is metabolized to nicotinic acid and a small amount is metabolized to serotonin via the intermediary stage of 5-hydroxy-tryptophan (5-HTP). The enzyme responsible for this step, tryptophan hydroxylase, is the rate-limiting enzyme for serotonin production and is normally only half saturated. The current use of TRPO 1 as an adjunct in the management of affective disorders utilizes this pathway to increase serotonin levels.

2.2 Tolerance In doses below 5 g/day, TRP01 may cause dry mouth and drowsiness. In higher doses (9 to 12 g/day), nausea, anorexia, dizziness and headache have been reported. Side effects disappear when medication is discontinued and in most cases only a light dizziness may persist. Sexual disinhibition has been reported in some patients with emotional disorders.

2.3 Toxicity In 1989, L-tryptophan from Japan was implicated in an outbreak of eosinophilia myalgia syndrome (EMS) in the United States, where it was sold over-the-counter as a dietary supplement14. A total of 37 deaths and 1500 cases of permanent disability were, after months of investigation, linked to the 13 Material for this section was taken from: Canadian Pharmacists Association, Compendium of Pharmaceuticals and Specialties, Webcom Ltd., Toronto, 1998; p. 1732-3.

14 Wilkins K; Wigle D. Can. Med. Assoc. J. (1990) 142: 1265-6.

consumption of the L-tryptophan produced by a single Japanese company, Showa Denko15. The toxicity was linked to 1,11-ethylidene-bis-(L-tryptophan), an impurity in the product which originated from Showa Denko's use of genetically modified bacteria. This impurity has never been noted to exist in L-tryptophan produced using conventional, non-genetically modified bacteria. Furthermore, unlike herbals and dietary supplements, prescription drugs are continually subjected to rigorous purity checks before allowing the drugs to be shipped to distributors, thus eliminating any chance of contamination.

The L-tryptophan used in this study will be produced and supplied by ICN
Pharmaceuticals Inc. of Montreal, Canada.

An increased incidence of bladder cancer has been observed in experimental animals after implantation of pellets containing any of the seven L-tryptophan metabolites formed by tryptophan pyrrolase. Elevated levels of these metabolites have been found in the urine of bladder cancer patients relative to controls, in patients who had a recurrence of cancer relative to those who did not, and in patients taking oral contraceptives or hormones. A large study performed by the National Cancer Institute, however, did not find L-tryptophan to produce cancer in either mice or rats.

Vitamin B6 has been reported to reduce L-tryptophan metabolites to normal levels, thereby suggesting its use in patients consuming many times the normal daily intake of L-tryptophan.

Xanthurenic acid, which is increased by high levels of L-tryptophan, has a diabetogenic action in animals, possibly due to its ability to bind insulin, recommending caution in the use of TRP01 in patients with a family history of diabetes.

Oral L-tryptophan was shown to cause pulmonary edema and emphysema in ruminants, mediated by bacterial conversion of L-tryptophan to skatole (3-methylindole). This is only of concern in humans when bacteria exist high in the gastrointestinal tract due to conditions such as achlorhydria or where TRP01 reaches the bacterial populations lower in the gastrointestinal tract due to malabsorption.

Animal studies suggest that photooxidation of L-tryptophan and some of its metabolites, such as kynurenine, may be involved in cataract formation.
Although there is no evidence that this occurs in humans, it is possible that TRP01 administration could raise lenticular L-tryptophan and kynurenine 15 Mayeno AN; Gleich GJ. TIBTECH (1994) 12: 346-352.

concentrations. This might make subjects more susceptible to cataract formation, particularly if exposed to ultraviolet light.

Patients taking high doses of TRP01 should not be protein deprived since an imbalance in amino acids can ensue.

2.4 Drug interactions Drug interactions between TRPOI and other CNS-affecting drugs have been reported. When TRP01 was given in combination with monoamine oxidase (MAO) inhibitors, a higher incidence of side effects was noted. The most common of these side effects were dizziness, nausea and headache. At a dosage of 20 to 50 mg/kg TRP01 in addition to MAO inhibitors, ethanol-like intoxication, drowsiness, hyperreflexia and clonus have been reported. Single case reports of adverse reactions to this drug combination include hypomanic behaviour, ocular oscillation, ataxia and myoclonus. Such symptoms resemble the "serotonin syndrome" seen in experimental animals, which consists of tremor, hypertonus, myoclonus and hyperreactivity. These symptoms disappear shortly after cessation of TRPO 1 and no detrimental long-term effects have been reported.

When TRPO1 was given in combination with fluoxetine, the following side effects have been reported: agitation, restlessness, poor concentration, nausea, diarrhea, and worsening of obsessive-compulsive disorder. Neither drug caused similar side-effects when administered alone.

TRPOI, when given with lithium, may increase some side effects associated with lithium therapy by potentiating the lithium effect (nausea, vomiting, dermatological eruptions, psoriasis, alopecia).

2.5 Safety TRPOI is an approved drug in Canada and has been prescribed by physicians since 1985. It has been available over the counter outside Canada for even longer. Other than the above mentioned epidemic of EMS caused by an impurity in one company's L-tryptophan, no serious adverse effects have been reported for the drug throughout its many years of use.

Previous clinical trials have tested TRP01 in patients with AD and have confirmed its safety for use in elderly demented patients16' 16 Bentham PW. lnt.'I Clin. Psychopharm. (1990) 5: 261-272.

17 Smith DF; Stromgren E; Petersen HN; Williams DG; Sheldon W. Acta Psychiatry. Scand. (1984) 70: 470-477.

3. STUDY OBJECTIVE

The primary objective of the study will be to evaluate any interruption in the progressive deterioration of cognitive abilities in patients with AD.

4. STUDY DESIGN

The clinical trial will be a double-blind, placebo-controlled study with patients being randomized in a 2:1 ratio into groups A and B, respectively:

Group A: TRP01 Ig b.i.d. for 26 weeks Group B: placebo capsules b.i.d. for 26 weeks After the initial screening, clinic visits will take place at 0, 13 and 26 weeks.
The blinding will be maintained until all patients have completed the 26-week trial. Any patients wishing to continue taking the medication upon completion of the trial will be provided a prescription for the drug and may purchase it at their own expense.

5. SELECTION OF STUDY POPULATION
5.1 Inclusion criteria Patients will be selected in order to fulfill both of the following definitions:
= Dementia according to DSM-IV criteria18:
- development of multiple cognitive deficits manifested by both memory impairment and one or more of the following cognitive disturbances:
aphasia, apraxia, agnosia, disturbance in executive functioning - the above-mentioned deficits cause significant impainnent in social or occupational functioning and represent a significant decline from a previous level of functioning - they do not occur exclusively during the course of delirium 18 American Psychiatric Association, Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV), Washington DC, 1994.

= Dementia according to communicative disorders and stroke criteria (NINCDS-ADRDA) ':
- dementia established clinically and documented by a Folstein Mini-Mental State Examination (MMSE)19 - deficits in two or more areas of cognition - progressive worsening of memory and other cognitive functions - no disturbance of consciousness - onset between 40 and 90 years of age - absence of systematic disorders or other brain diseases that in and of themselves can account for progressive deficit in memory and cognition (see exclusion criteria) Furthermore, patients must fulfill the following criteria:
= men, or postmenopausal or surgically sterilized women = aged 50 years or older = with severity of dementia of mild to moderate degree as reflected by a score of between 14 and 26, inclusive, on the MMSE
= with a minimum one-year duration of the symptomatology (progressive worsening of memory and other cognitive functions) = living at home or in an institution provided that they have reliable caregivers capable of attending each clinic visit, completing required evaluations, supervising and ensuring the administration of all doses of medication = presenting a reasonably good nutritional status = able to perform the required psychometric tests and evaluations. Visual and auditory acuity (with glasses or hearing aid if required) must be sufficient to complete the protocol-specified procedures.
= clinical laboratory battery (see section 7.2.2) must yield results within normal limits or determined as not clinically significant by the study physician for the patient's sex and age = patients and their substitute decision-makers must have signed the written informed consent form Evidence of diffuse atrophy, enlarged ventricles and widened sulci are characteristic findings in patients with AD and are acceptable, as well as the presence of one lacuna (subcortical hypodensity less than 15 mm diameter).
Mild white matter changes described as "Leukoaraiosis" are also acceptable.
5.2 Exclusion criteria Patients with any of the following will not be included in the study:

19 Rosen WG; Terry RD; Fuld P; Katzman R; Peck A. Ann. Neurol. (1979) 7:
486-8.

= Patients with any other cause of dementia as evidenced by medical history, general physical and neurological examination, laboratory tests, and neuroradiological findings:
- Vascular dementia, as evidenced by Modified Hachinski Ischemia Scale15 score > 4 - Depressive pseudementia, as evidenced by cognitive disturbances concomitant to a major depressive episode according to DSM-IV
and/or a history of more than one major depressive episode - DSM-IV criteria for any major psychiatric disorder including schizophrenia, alcohol or substance abuse - Huntington's chorea or Parkinson's disease, evidenced by neurological examination, with an onset prior to or concurrent with dementia - Creutzfeldt-Jakob disease - Intracranial mass lesion (e.g. cerebral neoplasm, subdural haematoma), normal pressure hydrocephalus (at cranial CT or MRI) - Clinically important head injury (i.e. with loss of consciousness) within one year of onset of dementia - History or current evidence of stroke - Onset of dementia following cardiac arrest or heart surgery - Neurosyphillis: patient with a positive rapid plasma reagin (RPR) should have the test repeated. If the result of the retest is positive, a Fluorescent Treponemal Antibody (FTA) titre must be performed. If the FTA is positive the patient will not be included - Seropositivity for HIV (specific testing should be performed in case of risk factors) - Vitamin B12 deficiency (i.e. serum Bi2 level < lower limit of the normal range) or folate deficiency (i.e. serum folate level < lower limit of the normal range). Patients with a B1Z or folate deficiency may be supplemented during the screening period. They may be included if B12 or folate plasma level is normalized and if functional status is considered as stable at the end of the screening period. It is important to exclude patients whose dementia symptoms result from such deficiencies - Uncorrected hypothyroidism (i.e. abnormal free T4, ultrasensitive TSH) = Patients with other relevant concomitant diseases:
- Patients with history or current evidence of a sleep disorder - Patients with a clinically significant cardiovascular, renal, hepatic, pulmonary (including severe asthma), gastrointestinal, endocrine, metabolic, opthalmologic, or hematologic condition - Current evidence or history within the past year of myocardial infarction (MI), congestive heart failure - Blood pressure at screening (on repeated measures) of> 180 mm Hg systolic or 100 mm Hg diastolic - Impaired renal, hepatic, or gastrointestinal function, which could interfere with drug absorption, metabolism or excretion - Seizure disorder - Diabetes - Earlier diagnosis or current evidence of cataracts - Achlorhydria - Progressive fatal disease (other than AD) - Treatment with any other investigational drug in the last 8 weeks prior to screening - Previous administration of TRPO1 5.3 Recruitment Study patients will be outpatients or institutionalized patients with a reliable caregiver attending the visits to the investigational center.

5.4 Number of subjects It is planned to enter 30 patients in the study, 20 of whom would receive TRP01 while the remaining 10 received placebo capsules.

6. STUDY DRUGS
6.1 Study medication The study medication will consist of identical capsules containing either 500 mg TRPOI or 500mg corn starch as placebo.

Medication will be administered orally using a twice-daily (b.i.d.) regimen over the course of the 26 week study. The doses, each consisting of two capsules, will be administered with a small amount of water and a carbohydrate snack 1 hour before breakfast and at bed-time every day. The medication is to be taken without protein-rich food in order to prevent absorption competition from dietary amino acids such as phenylalanine which are known to exhibit such behaviour'. In the event of a missed dose, patients and caregivers will be instructed not to "catch up" by taking an extra dose.
Rather, they will be instructed to record the missed dose on the dosage diary and continue with the planned schedule.

The treatment regimens are as follows:

= Patients in Group A: TRPO1 lg b.i.d. for 26 weeks.
= Patients in Group B: placebo lg b.i.d. for 26 weeks.
6.2 Drug supplies and accountability The study medication will be supplied by ICN Pharmaceuticals Inc. in bottles of 100 500mg capsules. Identical capsules will be manufactured for the placebo group, save that placebo will be substituted for the TRPO1.

At the 13-week and 26-week visits, any excess capsules will be counted in order to ensure that the stated compliance is accurate and all medication is accounted for. At the end of the study, any unused or excess medication will be returned to the investigator. The investigator will keep accurate records of dates of dispensing and returning study drugs against the patient number.

6.3 Assignment of treatments to patients The treatment allocation will take place after visit 0 for patients satisfying all the inclusion/exclusion criteria. Each of these eligible patients will be assigned a unique patient number which will be used to identify patients throughout the study. They will also be assigned a unique treatment number corresponding to either TRPO1 or placebo. Prior to screening, the treatment numbers will be randomly assigned to correspond to either TRP01 or placebo by a person external to the study. Two-thirds of the treatment numbers will designate treatment with TRPO1, one-third will designate the administration of placebo.

Patients will be given cards bearing their treatment number and the investigator's phone number and will be instructed to have access to the cards at all times. If medical reasons warrant, the investigator can then be contacted and the randomization broken in order to disclose the patient's treatment to the attending physician. In such a case, the patient may have to be excluded from the trial.

Patients who, after randomization, decline to participate in the study before administration of the first dose of study medication will keep their randomization number. The next patient enrolled will be given the next number.

6.4 Concomitant treatments All concomitant treatments will be recorded in the patients' records along with the reasons for the prescription, the dosage, the frequency and the dates of administration. Such treatments should be kept to a minimum during the study.

The following medications are prohibited for safety reasons or to prevent a false positive result. Patients taking any of the following medication but wishing to participate in the study will be allowed to do so following a 4 week wash-out period:
- investigational drugs for AD or for any other disorder - MAO inhibitors (pheneizine, tranylcypromine, noclobemide) - fluoxetine - paroxetine - sertraline - fluvoxamine - lithium - any acetyl-cholinesterase (AChE) inhibitor (e.g. donepezil) - ginkgo biloba - ginseng - vitamin E
- estrogen for more than hormonal replacement - aspirin at doses of> 650 mg/day - non-steroidal anti-inflammatory drugs (NSAIDs) for more than 30 consecutive days (allowed p.r.n.):

- diclofenac - flurbiprofen - etodolac - ibuprofen - indomethacin - ketoprofen - ketorolac tromethamine - naproxen - sulindac - oxaprozin - tolmetin - tiaprofenic acid - floctafenine - choline magnesium trisalicylate - mefenamic acid - diflunisal - piroxicam - magnesium salicylate - tenoxicam - nabumetone - fenoprofen 7. EVALUATION
7.1 Evaluation criteria 7.1.1. Tolerance criteria Clinical variables:
- reports of spontaneous adverse events - vital signs (blood pressure and heart rate both in the sitting and standing position) - physical examination including body weight, review of body systems and neurological evaluation (gait, speech, coordination, tremor, cranial nerves, muscle strength and tone, sensation and level of consciousness) Urine - glucose, occult blood, protein 7.1.2. Efficacy criteria - MMSE score - Alzheimer's Disease Assessment Scale, cognitive subpart (ADAS-Cog) as an evaluation of cognitive functioning - Clock test - Alzheimer's Disease Cooperative Study - Clinical Global Impression of Change (ADCS-CGIC) - Neuropsychiatric Inventory (NPI) - Disability Assessment for Dementia (DAD) - Functional Activities Questionnaire (FAQ) 7.1.3 Compliance criteria Compliance will be assessed by examining the dosage diary, questioning the caregiver and counting the capsules in the returned medication.

7.1.4 Other parameters The Pittsburgh Sleep Quality Index and Cornell Depression Index will be performed at visits 0, 2 and 3 to ensure that any change in a patient's dementia is unrelated to the reported sedative and anti-depressant properties of 7.2 Methods of evaluation 7.2.1 Efficacy The evaluation tools will be used as follows:
- MMSE: visits 0, 1, 2 and 3.
- ADAS-Cog: visits 1, 2 and 3 - Clock test: visits 1,2 and 3 - ADCS-CGIC: visits 1 and 3 - NPI: visits 1 and 3 - DAD: visits I and 3 - FAQ: visits I and 3 For each evaluation the patient will be called to the hospital with the caregiver.
In addition to the study investigator, a neuropsychologist and a research assistant are needed to administer the efficacy evaluations. The DAD, NPI
and FAQ will be administered by the neuropsychologist, and the ADAS-cog and clock test will be performed by a research assistant. The study investigator will perform the MMSE, ADCS-CGIC, physical and neurological examinations and will manage any adverse events.

ADAS-Cogzo The ADAS-Cog is a validated scale developed specifically as a research instrument to evaluate the severity of cognitive deficits and behavioural 20 Rosen WG; Mohs RC; Davis KL. American J. Psychiat. (1984) 141: 1356-64.

problems characteristic of patients with AD. This test can usually be administered to patients with AD in about 30 minutes.

The ADAS-Cog will be performed by a research assistant trained in the administration and scoring of the test. This individual will administer the test at each visit.

The ADAS-Cog is an objective 11-item test battery that relies solely on the patient's ability to perform specific tasks during the administration of the test.
Furthermore, it captures the cardinal features of AD. ADAS-Cog scores range from 0 to 70 points, with higher scores indicating a greater degree of cognitive impairment.

The MMSE is a short standard assessment instrument for diagnosing the presence and severity of cognitive impairment, monitoring the progression of the disease over time, and assessing potential therapeutic effects. The test will be administered by the study investigator in about 10 minutes.

The MMSE assesses 6 domains of cognitive functioning: orientation, registration, attention, recall, language and constructional abilities. The maximum score is 30 points, with lower scores indicating a greater degree of cognitive impairment.

The ADCS-CGIC is a global change measure intended for use in the setting of controlled clinical investigations to determine whether the effects of the investigational antidementia drug are of sufficient magnitude to allow their detection in an interview conducted by a clinician familiar with the manifestations of dementia. The ADCS-CGIC is intended to be based entirely upon information collected during an interview (approx. 30 minutes) with the patient. The study investigator will be responsible for interviewing and rating the patient throughout the course of the clinical investigation. This is critical because the ADCS-CGIC is intended to capture change occurring in a patient from the baseline interview to the time of the assessment. To prepare for the baseline interview, the ADCS-CGIC investigator should review all sources of information available about the patient, including but not limited to, the 21 Folstein MF; Folstein SE; McHugh PR. J. Psychiat. Res. (1975) 12: 189-198.

22 Schneider LS; Olin JT; Doody RS; Clark CM; Morris JC; Reisberg B;
Schmitt FA; Grundman M; Thomas RG; Ferris SH. Alzheimer Disease and Assoc. Disorders (1997) 11: S22-S32.

medical history, the results of physical examination, laboratory and special tests.
The ADCS-CGIC consists of a seven-point rating in which a rating of I means "very much improved", a rating of 4 is "no change" and a rating of 7 is "very much worse".

The NPI is an instrument developed to assess 10 behavioural disturbances occurring in patients with dementia: delusions, hallucinations, agitation/
aggression, dysphoria, anxiety, elation/euphoria, apathy, disinhibition, irritability/ lability and aberrant motor behaviour. The neuropsychologist will perform this test.

The objectives of the DAD Scale are to quantitatively measure functional abilities in the activities of daily living (ADL) in individuals with cognitive impairments such as dementia and to help delineate areas of cognitive deficits which may impair performance in ADL. The neuropsychologist will perform this assessment. It is preferable to perform the interview in a quiet environment alone with the caregiver. Basic and instrumental activities of daily living are examined in relation to executive skills to permit identification of problematic areas. The aim is to have a standardized, valid, reliable and sensitive measure of functional disability. This scale defines basic activities of daily living such as dressing, hygiene, continence and eating.

Instrumental activities include meal preparation, telephoning, housework, taking care of finance and correspondence, going on an outing, taking medications and ability to stay safely at home.

Clock test The clock test, administered by the research assistant, has the patient draw a clock showing 10:50 as the time. The test is out of 10 points, with marks allotted for drawing the circle, the placement and size of the numbers and for the placement and relative size of the big and little hands.

23 Cummings JL; Mega M; Gray K.; Rosenberg-Thompson S; Carusi DA;
Gornbein J. Neurology (1994) 44: 2308-2314.

24 Gauthier L.; Gauthier S; Gelinas T; Mclntyre M; Wood-Dauphines S.
Abstract: Sixth Congress of the International Psychogeriatric Association (1993) 9.

FA ZS
Like the DAD, the FAQ is a caregiver-based measure of functional activities.
The caregiver will be asked to provide performance ratings of the patient on complex, higher-order activities. Scores range from 0 to 30 with higher scores indicating greater impairment. The test will be administered by the study investigator in about 10 minutes.

7.2.2. Tolerance and safety Clinical variables:
- Adverse events reported by the patient or the caregiver, in reply to an open question "how have you been feeling since the last visit" will be recorded at each visit other than visit 0 - Vital signs will be monitored at visits 0, 2 and 3, including blood pressure and heart rate both in sitting and standing positions. Blood pressure will be measured by the nurse coordinator using a conventional sphygmomanometer - Physical examination including neurological evaluation will be conducted at visits 0 and 3 Urinalysis:
Performed at visits 0 and 3 7.2.3. Compliance This will be evaluated at each visit during the treatment period by - Examining the dosage diary - Questioning the caregiver - Counting the number of remaining capsules returned by the patient 7.2.4. Other parameters - Cornell Depression Index26 to evaluate any change in depression - Pittsburgh Sleep Quality Index27to detect any improvement in sleep 25 Pfeiffer RI; Kurosaki TT; Harrah CH. J. Gerontol. (1982) 37: 323-329.

26 Alexopoulos GS; Abrams RC; Young RC; Shamoian CA. Biol. Psychiatry (1988) 23: 271-284.

27 Buysse DJ; Reynolds CF; Monk TH; Berman SR; Kupfer DJ. Psychiatry Research (1989) 28: 193-213.

8. STUDY PROCEDURE

8.1 Pre-treatment Patients judged by the investigator to be likely candidates for the study will be contacted, informed about the study and invited to be tested for eligibility.
After discussion with family, substitute decision-maker and caregiver, interested patients can make appointments for their initial visit (visit 0).

Visit 0 (week -4 to 0) This visit will determine whether the patient is eligible for the study. The following steps will first be performed:
- check inclusion and exclusion criteria - explain the study to the patient, substitute decision-maker and caregiver.
Give them the information sheet and obtain their signatures on the written consent form The following data will be collected or checked:
- demographic data (including educational and occupational levels) - medical and familial history (familial history of dementia) - concomitant medication - previous medication (within the last three months) - physical examination (including weight) and vital signs - neurologic examination - MMSE and modified Hachinski score19 - Cornell Depression IndexZ6 - Pittsburgh Sleep Quality Index27 - urinalysis Patients failing to meet all the requirements for the above tests will be informed that they are ineligible for the study. Eligible patients will be assigned a patient number (first available number in sequential order) and will make an appointment for Visit 1. Patients taking prohibited medication will schedule their visit to follow a 4-week washout period.

8.2 Treatment Visit I (week 0) - baseline assessments: perform MMSE, ADAS-Cog, Clock test, ADCS-CGIC, NPI, ask the patient's caregiver to complete the DAD and FAQ
- record any concomitant medication - give the patient his/her treatment number and dispense study medication - make an appointment for visit 2 Visit 2 (week 13) - recover unused medication and complete the dosage record - record adverse events (open question to patient and caregiver) - review concomitant medications (note any changes since last visit) - record vital signs - conduct ADAS-Cog, Clock test, MMSE, Pittsburgh Sleep Quality Index and Cornell Depression Index - dispense study medication (with the corresponding treatment number) - make an appointment for visit 3 Visit 3 (week 26) - recover unused medication and complete the dosage record - record adverse events (open question to patient and caregiver) - review concomitant medications (note any changes since last visit) - record vital signs - perform physical examination including weight and neurological evaluation - conduct ADAS-Cog, Clock test, MMSE, ADCS-CGIC, NPI, DAD, FAQ, Pittsburgh Sleep Quality Index and Cornell Depression Index - urinalysis 8.3 Post treatment Any patients wishing to continue treatment at the end of the study will be provided with prescriptions for TRP01 and may obtain the drug at their own cost.

An effort should be made to obtain information on safety two weeks after the last drug intake. This safety follow-up should be documented in writing in the follow-up status form.

9. ADVERSE EVENTS
9.1 Definition The term adverse event covers any undesirable experience including intercurrent events (or diseases), drug reactions and clinical abnormalities or clinically significant laboratory test abnormalities which occur during the course of the trial.

9.2 Period of observation The period of observation for adverse events extends from the time the patient gives his/her informed consent, until he/she undergoes the final clinical examination scheduled in the study.

Adverse events occurring after this period of observation are to be reported if the investigator feels that there is a causal relationship to the test medication.
9.3 Procedures to be followed All adverse events observed by the investigator or reported by the patient whether or not considered related to the test medication will be followed up by the investigator until resolution, stabilization or loss to follow up.

All adverse events will be reported using the adverse event forms of the study book and assessed by the investigator in terms of severity and of relationship to the test medications. The outcome of the adverse event as well as the measures for this event will be described in the adverse event form of the study book.

9.4 Specific case of serious adverse events Definition:
An adverse event is considered to be serious if it:
- is fatal or life-threatening - suggests a significant hazard - is significantly or permanently disabling - requires or prolongs hospitalization - results from overdosage Furthermore, all cancers diagnosed during the course of the study will be considered as serious events.

Hospitalization for respite care is not considered to be a serious adverse event in this trial.

Period ofobservation For serious adverse events the period of observation extends until two weeks after the last day of treatment.

Medical follow-up of any serious adverse event or significantly abnormal laboratory determination will continue until the abnormality resolves, or until an adequate medical explanation is available.

Any serious adverse event will be recorded using a serious adverse event form and will include the following information:
- the identification of the patient (initials, patient number in the study, treatment number, date of birth, sex) - the treatment given, the dates of the beginning and the end of this treatment - a description of the adverse event and its date of onset - the measures undertaken to treat the adverse event - the opinion of the investigator as to the relationship of the event to the test medication, if possible at this stage - the signature of the investigator Experience has shown that the storage of a plasma sample taken at the time of onset of the serious adverse event can often later greatly assist in the assessment of etiology. Therefore, if possible, 5 mL of blood should be collected in a heparinised tube. After centrifugation, the plasma should be placed in a dry tube, labelled (patient number, initials, date and time of sampling) and frozen as quickly as possible. This frozen sample will be retained. If the sample is not required for testing, it wilt be discarded at the conclusion of the study.

The Ethics Committee will be informed by the investigator of serious unexpected adverse events occurring during the trial likely to affect the safety of trial subjects or conduct of the trial.

10. WITHDRAWALS AND DEFAULTERS
Reasons for withdrawal:
Patients may be withdrawn for the following reasons:
- at their own request - at the discretion of the investigator Patients will be withdrawn under the following circumstances:
- develops any significant intercurrent illness - requires treatment with a prohibited medication - fails to comply with the medication regimen such as more than 7 consecutive days of dosing between clinic visits In all cases, the reasons why patients are withdrawn will be recorded in detail in the Case Report Form and in the patient's medical records. Even if it is at the patient's own request, the reason for withdrawal will be carefully documented as it may be related to safety.

All efforts will be made to contact patients lost to follow-up (letter, telephone).
These efforts will be documented in the Case Report Form.

As far as possible, patients stopping treatment prior to completion of the study will be fully evaluated at projected week 26 visit.

Patient substitution Patients who do not complete the drug trial will not be replaced.
11. COLLECTION AND ANALYSIS OF DATA

11.1 Case Report Forms A Case Report Form will be used for each patient in the trial.

All forms will be filled in legibly in black ball point pen. All entries, corrections and alterations are to be made by the responsible investigator, psychologist, nurse coordinator or research assistant only. Corrections of data on the Case Report Form can be made only by crossing out the incorrect entry and putting the correct figure by the side. The incorrect figure will remain visible and the correction will be initialled and dated by the person making the correction.

A reasonable explanation will be given on the CRF by the investigator for any missing data.

11.2 Number of patients It is planned to enter a total of 30 patients. This trial is designed to give preliminary and exploratory results as to the interruption of the progressive deterioration of cognitive abilities in patients with Alzheimer's disease.
There is hence no statistical basis for this sample size which is obviously insufficient to detect minor improvements in patients taking TRPO 1 relative to those taking placebo.

11.3 Statistical methods 11.3.1 Data handling procedures All data, including all the information on CRFs, will be entered into a secure computer database.
11.3.2 Statistical analysis The data from the trial will be summarized with adequate descriptive statistics.
1. Statistical description of the general and medical characteristics and the baseline measurements of efficacy parameters will be provided for the overall population and for the two randomized groups using appropriate statistics (i.e. frequency, mean, median, standard deviations).

2. Study conduct: a complete description of exposure to trial drugs in terms of dosages and durations will be presented. Data will be examined for protocol compliance. Counts of patients who discontinued the trial, overall and by cause, will be presented in total and by time interval.

3. Analysis of the efficacy parameters: the medium term assessment of efficacy will only include the data from patients who complete the trial.

Statistical comparisons will be made between the group taking TRPO1 and that taking placebo, in terms of the scores obtained on the ADAS-Cog, Clock test, ADCS-CGIC, DAD, NPI, FAQ and MMSE tests.

4. Analyses of tolerance data:
General: Tolerance will be assessed on the population of patients enrolled in the trial and who are exposed to TRP01 (intake of at least one dose of TRP01).
Adverse events: AEs reported during the trial will be recorded in patients' files. Frequency will be displayed by body-system and by type of AE within the body-system. The following frequency tables will be presented: overall frequency, frequency of patients with severe AEs, frequency of patients with related AEs, frequency of related serious AEs, frequency of patients with AEs leading to study discontinuation. The estimates of AE frequencies will be carried out on treatment emergent signs or symptoms (TESS). TESS are adverse events which appeared after the start of the study treatment or which are present in the pre-selection phase but worsened under study treatment.
Clinical tolerance: Descriptive statistics on vital signs and frequency of abnormalities will be displayed for each time point.

12. ETHICAL AND LEGAL ASPECTS

The study will be conducted using TRPO1, an agent which is an approved drug in Canada.

12.1 Declaration of Helsinki The study will be performed in accordance with the guidelines of the Declaration of Helsinki, Hong Kong Amendment 1989.

12.2 Patient information and informed consent Before being admitted to the clinical study the patient will have consented to participate after he/she has had the nature, scope and schedule of the clinical study and possible consequences of the treatment explained in a form that is understandable to her/him. The patient will also be given an information sheet about the study.

The patient's consent form will be signed by the patient's caregiver because he/she will be an active participant in the study (takes patient to the visits, completes tests). The consent form will also be signed by the patient's substitute decision-maker (if other than the caregiver).

The patient's consent will be confirmed by the signature of the investigator.
The patient, substitute decision-maker and caregiver will sign 3 copies of the consent. One copy will be given to the patient/caregiver and another to the substitute decision-maker. The third copy of each patient's signed informed consent will be stored by the investigator in the study file until the end of the study. Then the investigator will archive these documents for 15 years.

12.3 Ethics committee (EC) approval/information Before the start of the study, the protocol, the consent form, any amendments (where appropriate), and other appropriate documents will be submitted to the Queen's University Ethics Committee.

Before inclusion of the first patient in the study, the investigator will have received the written EC approval of the protocol.

The EC will be informed of all serious unexpected adverse events likely to affect the safety of trial subjects or the conduct of the trial.

12.4 Confrdentiality All patient names will be kept confidential. Patients will be identified throughout documentation and evaluation by the number allocated to them during the study. The patients will be told that all study findings will be stored on computer and handled in strictest confidence.

13. PROTOCOL AMENDMENTS - PREMATURE DISCONTINUATION
OF STUDY

13.1 Protocol amendments The investigator will not alter this study protocol without obtaining the agreement of the EC. Amendments will only be made in exceptional cases once the study has started. Changes will be agreed to in writing and signed by the investigator and the EC. The changes then become part of the study protocol.

13.2 Premature discontinuation The investigator has the right to discontinue this study at any time for medical and/or administrative reasons. In such a case, the EC will be informed immediately.

14. USE OF INFORMATION AND PUBLICATION

The findings of this study will be published in a scientific journal and may be presented at a scientific meeting. Patient names will not be released in any communication concerning the trial.

15. TIME TABLE

The schedule dates are:
- first patient screened: April 18, 2001 - last patient out: June 1, 2002 16. DECLARATION OF TRIAL PERSONNEL

I do not have any financial investment in ICN Pharmaceuticals Inc., the producer and supplier of TRP01.

Investigator and Study Manager (place, date) Donald Weaver Study Neuropsychologist (place, date) Jill Irwin Nurse Coordinator (place, date) Sandy Weatherby Research Assistant (place, date) Michael Carter 17. DECLARATION OF INVESTIGATOR

The information contained in this study protocol is consistent with:
- the current risk/benefit evaluation of the test drug - the moral, ethical and scientific principles governing clinical research as set out in the Declaration of Helsinki and good clinical practice guidelines I have read this study protocol and agree that it contains all the information required to conduct the study. I agree to conduct the study as set out in this protocol.

The first patient will not be included in the study until approval has been received from the Queen's University Ethics Committee.

The study will be conducted in accordance with the moral, ethical and scientific principles governing clinical research as set out in the Declaration of Helsinki.

I agree to obtain, in the manner described in this study protocol, written informed consent to participate for all patients in this study.

I am aware of the requirements for the correct reporting of serious adverse events and I undertake to document and to report such events as requested.
Investigator and Study Manager (place, date) Donald Weaver

Claims (88)

1. A method for treating a protein folding disorder comprising administering a compound of formula (I) to a subject:

wherein A and B are independently a mono- or bicyclic aromatic or heteroaromatic substituent; wherein n=0 or 1; wherein, when n=1, R1 and R 2 are independently hydrogen, alkyl, cycloalkyl, alkoxy, hydroxy, halogen, or aryl;
wherein A is substituted by A1(x) and B is substituted by B1(y) ; wherein x and y are independently an integer from 0 to 4; and A1(x) and B1(y) are each independently, for each value of x and y, selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, thio, thioether, cyano, nitro, halogen, carboxylic acid and sulfonic acid;

or a pharmaceutically acceptable salt thereof;

wherein the subject is treated for the protein folding disorder.

2. The method of claim 1, wherein A and B are independently selected from the group consisting of phenyl, pyridyl, pyrrolyl, thiophenyl, furanyl, triazolyl, indolyl, naphthyl, benzofuranyl, quinolinyl and isoquinolinyl.

3. The method of claim 2, wherein at least one of A and B are indolyl.

4. The method of claim 3, wherein both A and B are indolyl.

5. The method of claim 4, wherein the compound of formula (I) is:

6. The method of claim 5, wherein x is 1 and A1 is at the 5, 6 or 7 position.

7. The method of claim 6, wherein x is 1 and A1 is CO2H.

8. The method of claim 5, wherein B1 is at the 5 or 6 position.

9. The method of claim 8, wherein B1 is selected from the group consisting of halogen, alkyl, alkoxy, aryl, thio, thioether, and trihalomethoxy.

10. The method of claim 4, wherein the compound of formula (I) is:

11. The method of claim 10, wherein x is 1 and A1 is at the 5 position;
wherein R1 and R2 are independently hydrogen, alkyl, cycloalkyl, alkoxy, hydroxy, halogen, or aryl.

12. The method of claim 11, wherein x is 1 and A1 is CO2H.

13. The method of claim 10, wherein B1 is at the 5 or 6 position.

14. The method of claim 13, wherein B1 is selected from the group consisting of halogen, alkyl, alkoxy, aryl, thio, thioether, and trihalomethoxy.

15. The method of claim 4, wherein the compound of formula (I) is:

(I)

16. The method of claim 15, wherein x is 1 and A1 is at the 5 position;
wherein R1 and R2 are independently hydrogen, alkyl, cycloalkyl, alkoxy, hydroxy, halogen, or aryl.

17. The method of claim 16, wherein x is 1 and A1 is CO2H.

18. The method of claim 15, wherein B1 is at the 5 or 6 position.

19. The method of claim 18, wherein B1 is selected from the group consisting of halogen, alkyl, alkoxy, aryl, thio, thioether, and trihalomethoxy.

20. The method of claim 15, wherein y is 1 and B 1 is at the 5 position;
wherein R1 and R2 are independently hydrogen, alkyl, cycloalkyl, alkoxy, hydroxy, halogen, or aryl.

21. The method of claim 20, wherein y is 1 and B1 is CO2H.

22. The method of claim 21, wherein A1 is at the 5 or 6 position.

23. The method of claim 22, wherein A1 is selected from the group consisting of halogen, alkyl, alkoxy, aryl, thio, thioether, and trihalomethoxy.

24. The method of claim 1, wherein the compound of formula I is selected from the group consisting of:

3,3'-bis-indolyl;
5-methoxy-3-(5-methoxy-indol-3 -yl)-indole;
3-(5-bromo-indol-3-yl)-indole-5-carboxylic acid;
3-(indol-3-yl)-indole-5-carboxylic acid;
3-(5-methoxy-indol-3-yl)-indole-5-carboxylic acid;
3-(5-fluoro-indol-3-yl)-indole-5-carboxylic acid;
3-(5-chloro-indol-3-yl)-indole-5-carboxylic acid;
3-(5-methyl-indol-3-yl)-indole-5-carboxylic acid;
3-(5-(trifluoromethoxy)-indol-3-yl)-indole-5-carboxylic acid;
3-(indol-3-yl)-indole-6-carboxylic acid; and a pharmaceutically acceptable salt thereof.

25. The method of claim 1, wherein the compound of formula I is selected from the group consisting of:

di-(indol-3-yl)methane;
3-((indol-1-yl)methyl)-indole;
bis(5-methoxy-indol-3-yl)methane;
5-methoxy-3-((5-methoxy-indol-1-yl)methyl)-indole;
3-((indol-3-yl)methyl)-indole-5-carboxylic acid;
bis-(indole-5-carboxylic acid-3-yl)methane;
bis-(5-hydroxy-indol-3-yl)methane;
1,2-di-(indol-3-yl)ethane;

3-(2-(5-bromo-indol-3-yl)ethyl)-indole-5-carboxylic acid;

1,2-bis-(indole-5-carboxylic acid-3-yl)ethane; and a pharmaceutically acceptable salt thereof.

26. The method of any of claims 1-25, wherein the protein folding disorder being treated is a neurodegenerative disease.

27. The method of claim 26, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, dementia, Parkinson's disease, Huntington's disease, prion-based spongiform encephalopathy and a combination thereof.

28. The method of claim 26, wherein the neurodegenerative disease is Alzheimer's disease.

29. The method of any of claims 1-25 wherein the compound attenuates an increase in thioflavin T fluorescence by greater than 30% at 20 hours, relative to beta-amyloid with vehicle as a control, in a beta-amyloid thioflavin T aggregation assay.

30. The method of any of claims 1-25 wherein the compound attenuates an increase in thioflavin T fluorescence by greater than 60% at 20 hours, relative to beta-amyloid with vehicle as a control, in a beta-amyloid thioflavin T aggregation assay.

31. The method of any of claims 1-25 wherein the compound attenuates an increase in thioflavin T fluorescence by greater than 90% at 20 hours, relative to beta-amyloid with vehicle as a control, in a beta-amyloid thioflavin T aggregation assay.

32. The method of any of claims 1-25 wherein the compound attenuates an increase in thioflavin T fluorescence by greater than 30% at 30 hours, relative to beta-amyloid with vehicle as a control, in a beta-amyloid thioflavin T aggregation assay.

33. The method of any of claims 1-25 wherein the compound attenuates an increase in thioflavin T fluorescence by greater than 60% at 30 hours, relative to beta-amyloid with vehicle as a control, in a beta-amyloid thioflavin T aggregation assay.

34. The method of any of claims 1-25 wherein the compound attenuates an increase in thioflavin T fluorescence by greater than 90% at 30 hours, relative to beta-amyloid with vehicle as a control, in a beta-amyloid thioflavin T aggregation assay.

35. The method of any of claims 1-25 wherein beta-amyloid, when co-incubated with the compound, exhibits circular dichroism, at 193nm after 48 hours, of less than that of beta amyloid with vehicle.

36. The method of any of claims 1-25 wherein beta-amyloid, when co-incubated with the compound, exhibits circular dichroism, at 193nm after 72 hours, of less than that of beta amyloid with vehicle.

37. The method of any of claims 1-25 wherein beta-amyloid, when co-incubated with the compound, exhibits circular dichroism, at 193nm after 48 hours, of at least 2 mdeg less than that of beta amyloid with vehicle.

38. The method of any of claims 1-25 wherein beta-amyloid, when co-incubated with the compound, exhibits circular dichroism, at 193nm after 72 hours, of at least 2 mdeg less than that of beta amyloid with vehicle.

39. The method of any of claims 1-25 wherein beta-amyloid, when co-incubated with the compound, exhibits circular dichroism, at 193nm after 72 hours, of at least 5 mdeg less than that of beta amyloid with vehicle.

40. A method of treating a protein folding disorder comprising administering to a patient in need thereof an effective amount of a compound which attenuates the increase in thioflavin T fluorescence by greater than 30%, relative to beta-amyloid with vehicle as a control, at 20 hours when subjected to a beta-amyloid thioflavin T
aggregation assay.

41. A method of treating a protein folding disorder comprising administering to a patient in need thereof an effective amount of a compound which attenuates the increase in thioflavin T fluorescence by greater than 60%, relative to beta-amyloid with vehicle as a control, at 20 hours when subjected to a beta-amyloid thioflavin T
aggregation assay.

42. A method of treating a protein folding disorder comprising administering to a patient in need thereof an effective amount of a compound which attenuates the increase in thioflavin T fluorescence by greater than 90%, relative to beta-amyloid with vehicle as a control, at 20 hours when subjected to a beta-amyloid thioflavin T
aggregation assay.

43. A method of treating a protein folding disorder comprising administering to a patient in need thereof an effective amount of a compound which attenuates the increase in thioflavin T fluorescence by greater than 30%, relative to beta-amyloid with vehicle as a control, at 30 hours when subjected to a beta-amyloid thioflavin T
aggregation assay.

44. A method of treating a protein folding disorder comprising administering to a patient in need thereof an effective amount of a compound which attenuates the increase in thioflavin T fluorescence by greater than 60%, relative to beta-amyloid with vehicle as a control, at 30 hours when subjected to a beta-amyloid thioflavin T
aggregation assay.

45. A method of treating a protein folding disorder comprising administering to a patient in need thereof an effective amount of a compound which attenuates the increase in thioflavin T fluorescence by greater than 90%, relative to beta-amyloid with vehicle as a control, at 30 hours when subjected to a beta-amyloid thioflavin T
aggregation assay.

46. A method of treating a protein folding disorder comprising administering to a subject in need thereof an effective amount of a compound which, when co-incubated with beta-amyloid, causes the peptide to exhibit circular dichroism, at 193nm after 48 hours, of less than that of beta amyloid with vehicle.

47. A method of treating a protein folding disorder comprising administering to a subject in need thereof an effective amount of a compound which, when co-incubated with beta-amyloid, causes the peptide to exhibit circular dichroism, at 193nm after 48 hours, of at least 2 mdeg less than that of beta amyloid with vehicle.

48. A method of treating a protein folding disorder comprising administering to a subject in need thereof an effective amount of a compound which, when co-incubated with beta-amyloid, causes the peptide to exhibit circular dichroism, at 193nm after 72 hours, of less than that of beta amyloid with vehicle.

49. A method of treating a protein folding disorder comprising administering to a subject in need thereof an effective amount of a compound which, when co-incubated with beta-amyloid, causes the peptide to exhibit circular dichroism, at 193nm after 72 hours, of at least 2 mdeg less than that of beta amyloid with vehicle.

50. A method of treating a protein folding disorder comprising administering to a subject in need thereof an effective amount of a compound which, when co-incubated with beta-amyloid, causes the peptide to exhibit circular dichroism, at 193nm after 72 hours, of at least 5 mdeg less than that of beta amyloid with vehicle.

51. The method of any of claims 29-46, wherein the protein folding disorder being treated is a neurodegenerative disease.

52. The method of claim 46, wherein the neurodegenerative disease is selected from the group consisting of tauopathies, cerebral amyloid angiopathy, Lewy body diseases, Alzheimer's disease, dementia, Huntington's disease, prion-based spongiform encephalopathy and a combination thereof.

53. The method of claim 48, wherein the neurodegenerative disease is Alzheimer's disease.

54. The method of any of claims 1-25 wherein the aromatic substituents of the compound each exhibit sufficient gas-phase cation-n binding energy to cationic residues of the protein to treat the protein folding disorder.

55. The method of any of claims 1-25 wherein the aromatic substituents of the compound each exhibit gas-phase cation-it binding energy to cationic residues of the protein of at least 15 kcal/mol in a RHF/6-31G(d)//RHF/3-21G calculation, as implemented within the Gaussian98 computer program.

56. A method of treating a protein folding disorder comprising administering to a patient in need thereof, a compound whose aromatic or heteroaromatic substituents each exhibit sufficient gas-phase cation-.pi. binding energy to cationic residues of the protein to treat the protein folding disorder.

57. A method of treating a protein folding disorder comprising administering to a patient in need thereof, a compound whose aromatic or heteroaronatic substituents each exhibit a gas-phase cation-.pi. binding energy to cationic residues of the protein of at least 15 kcal/mol in a RHF/6-31G(d)//RHF/3-21G optimization calculation, as implemented within the Gaussian98 computer program.

58. The method of any of claims 54-57, wherein the protein folding disorder being treated is a neurodegenerative disease.

59. The method of claim 58, wherein the neurodegenerative disease is selected from the group consisting of tauopathies, cerebral amyloid angiopathy, Lewy body diseases, Alzheimer's disease, dementia, Huntington's disease, prion-based spongiform encephalopathy and a combination thereof.

60. The method of claim 58, wherein the neurodegenerative disease is Alzheimer's disease.

61. A pharmaceutical composition comprising pharmaceutically acceptable excipients and an effective amount of a compound of any of claims 1-25 to treat a protein folding disorder.

62. The pharmaceutical composition of claim 61, wherein the protein folding disorder being treated is a neurodegenerative disease.

63. The pharmaceutical composition of claim 62, wherein the neurodegenerative disease is selected from the group consisting of tauopathies, cerebral amyloid angiopathy, Lewy body diseases, Alzheimer's disease, dementia, Huntington's disease, prion-based spongiform encephalopathy and a combination thereof.

64. The pharmaceutical composition of claim 63, wherein the neurodegenerative disease is Alzheimer's disease.

65. A method for treating a protein folding disorder comprising administering a compound of formula (I) to a subject:

wherein A and B are independently a mono- or bicyclic aromatic or heteroaromatic substituent; wherein n is an integer from 1 to 10; wherein, when n is not 0, R1 and R2 are substituted at each of one or more carbons and are independently hydrogen, alkyl, cycloalkyl, alkoxy, hydroxy, halogen, or aryl; wherein A is substituted by A1(x) and B is substituted by B1(y); wherein x and y are independently an integer from 0 to 4; and A1(x) and B1(y) are each independently, for each value of x and y, selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, thio, thioether, cyano, nitro, halogen, carboxylic acid and sulfonic acid;

or a pharmaceutically acceptable salt thereof;

wherein the subject is treated for the protein folding disorder.

66. The method of claim 66, wherein n is 0 or 1.

67. A compound of formula (IX):

wherein A and B are indolyl substituents; wherein A is substituted by A1(x) and B is substituted by B1(y); wherein x and y are independently an integer from 0 to 3 and the sum of x and y is from 1 to 3; and each A1 and each B1 are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, thio, thioether, cyano, nitro, halogen, carboxyl and sulfonic acid;
or a pharmaceutically acceptable salt thereof.

68. A compound of formula (IX):

wherein A and B are indolyl substituents; wherein A is substituted by A1(x) and B is substituted by B1(y); wherein x and y are independently an integer from 0 to 4 and the sum of x and y is at least 1;
each A1 and each B1 are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, thio, thioether, cyano, nitro, halogen, carboxyl and sulfonic acid;
or a pharmaceutically acceptable salt thereof.

69. A compound of formula (IX):

wherein A and B are indolyl substituents; wherein A is substituted by A1(x) and B is substituted by B1(y); wherein x and y are independently an integer from 0 to 4 and the sum of x and y is at least 1;
each A1 and each B1 are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, thio, thioether, cyano, nitro, halogen, carboxyl and sulfonic acid; provided that the total number of hydroxy substituents are less than 4;
or a pharmaceutically acceptable salt thereof.

70. A pharmaceutical composition comprising a compound of any of claims 67-69, and a pharmaceutically acceptable excipient.

71. A method for treating a protein folding disorder comprising administering a compound of any of claims 67-69 to a subject.

72. A compound of formula (X):

wherein A and B are indolyl substituents; wherein A is substituted by A1(x) and B is substituted by B1(y); wherein x and y are independently an integer from 0 to 4 and the sum of x and y is at least 1, provided that when x and y both equal 1, Al and B, are not both CO2H and are not both halogen;
p is 1 or 2; and each A1 and each B1 are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, thio, thioether, cyano, nitro, halogen, carboxyl and sulfonic acid;
or a pharmaceutically acceptable salt thereof.

73. A compound of formula (X):

wherein A and B are indolyl substituents; wherein A is substituted by A1(x) and B is substituted by B1(y); wherein x and y are independently an integer from 0 to 4 and the sum of x and y is at least 1;
p is 1 or 2; and each A1 and each B1 are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, thio, thioether, cyano, nitro, and sulfonic acid;
or a pharmaceutically acceptable salt thereof.

74. A compound of formula (X):

wherein A and B are indolyl substituents; wherein A is substituted by A1(x) and B is substituted by B1(y); wherein x and y are independently an integer from 0 to 4 and the sum of x and y is at least 1;

p is 1 or 2; and each A1 and each B1 are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, thio, thioether, cyano, nitro, halogen, carboxyl and sulfonic acid; provided that the total number of CO2H substituents are not more than 1 and the total number of halogen substituents are not more than 1;
or a pharmaceutically acceptable salt thereof.

75. A compound of formula (IX):

wherein A and B are indolyl substituents; wherein A is substituted by A1(x) and B is substituted by B1(y); wherein x and y are independently an integer from 0 to 4 and the sum of x and y is at least 1;
p is 2; and each A1 and each B1 are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, alkoxy, trihalomethoxy, aryloxy, arylcarbonyl; alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, thio, thioether, cyano, nitro, halogen, carboxyl and sulfonic acid;
or a pharmaceutically acceptable salt thereof.

76. A pharmaceutical composition comprising a compound of any of claims 72-75, and a pharmaceutically acceptable excipient.

77. A method for treating a protein folding disorder comprising administering a compound of any of claims 72-75 to a subject.

78. A compound of any of formula (III), (IV), (V), (VI), (VII) or (VIII) as disclosed herein, or a pharmaceutically acceptable salt thereof.

79. A pharmaceutical composition comprising a compound of claims 78, and a pharmaceutically acceptable excipient.

80. A method for treating a protein folding disorder comprising administering a compound of claim 78 to a subject.

81. A method for inhibiting tau protein aggregation comprising administering to a subject a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X) as disclosed herein, or a pharmaceutically acceptable salt thereof, to a subject

82. A method for the treatment of a subject having a protein folding disorder comprising administering an effective amount of a therapeutic agent to said patient, wherein said therapeutic agent binds to at least one of a BXBB, BBXB, AXBBXB
or BXBBXA receptor site of a protein associated with the protein folding disorder, and wherein said therapeutic agent is capable of binding to the 1AMB portion of the PDB
Structure of the HHQK region of an Amyloid Beta protein with a greater binding energy than -54.4 kcal/mol at the His 13-His 14 region.

83. A method for the treatment of a subject having a protein folding disorder comprising administering an effective amount of a therapeutic agent to said patient, wherein said therapeutic agent binds to at least one of a BXBB, BBXB, AXBBXB
or BXBBXA receptor site of a protein associated with the protein folding disorder, and wherein said therapeutic agent has a binding energy to the HHQK region of A.beta. that is 2% greater than the binding energy of L-tryptophan to the HHQK region of A.beta..

84. A composition for use in the treatment of a protein folding disorder, wherein said composition comprises a therapeutic agent capable of binding to at least one of a BXBB, BBXB, AXBBXB or BXBBXA receptor site of a protein associated with the protein folding disorder, and wherein said therapeutic agent is capable of binding to the 1AMB portion of the PDB Structure of the HHQK region of an Amyloid Beta protein with a greater binding energy than -54.4 kcal/mol at the His 13-His 14 region.

85. A composition for use in the treatment of a protein folding disorder, wherein said composition comprises a therapeutic agent capable of binding to at least one of a BXBB, BBXB, AXBBXB or BXBBXA receptor site of a protein associated with the protein folding disorder, and wherein said therapeutic agent has a binding energy to the HHQK region of A.beta. that is 2% greater than the binding energy of L-tryptophan to the HHQK region of A.beta..

86. A method of treating a protein folding disorder comprising administering to a patient in need thereof an effective amount of a compound which attenuates the increase in thioflavin T fluorescence by greater than 30%, relative to beta-amyloid with vehicle as a control, at 20 hours when subjected to a beta-amyloid thioflavin T
aggregation assay.

87. A method of treating a protein folding disorder comprising administering to a patient in need thereof, a compound whose aromatic or heteroaromatic substituents each exhibit sufficient gas-phase cation-.pi. binding energy to cationic residues of the protein to treat the protein folding disorder.

88. A method of treating a protein folding disorder comprising administering to a patient in need thereof, a compound whose aromatic or heteroaronatic substituents each exhibit a gas-phase cation-.pi. binding energy to cationic residues of the protein of at least 15 kcal/mol in a RHF/6-31G(d)//RHF/3-21G optimization calculation, as implemented within the Gaussian98 computer program.